Compositions and methods for delivery of immune cells to treat un-resectable or non-resected tumor cells and tumor relapse

ABSTRACT

The present disclosure provides compositions and methods for the delivery of immune cells to treat un-resectable or non-resected tumor cells and tumor relapse. The compositions comprise (i) a structure comprising an injectable polymer or scaffold comprising pores; (ii) lymphocytes disposed within the structure, (iii) at least one lymphocyte-adhesion moiety associated with the structure; and (iv) at least one lymphocyte-activating moiety associated with the structure, and optionally an immune stimulant.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/760,695, filed Sep. 4, 2015, which is a National Phase Application ofInternational Application No. PCT/US2014/011526, filed Jan. 14, 2014,which claims priority to and the benefit of U.S. Provisional ApplicationNos. 61/900,922 filed Nov. 6, 2013 and 61/752,423 filed Jan. 14, 2013.The entire contents of each of these applications are incorporated byreference herein.

FIELD OF THE DISCLOSURE

The present disclosure provides compositions and methods for thedelivery of immune cells to treat un-resectable or non-resected tumorcells and tumor relapse. The compositions comprise (i) a structure; (ii)lymphocytes, (iii) lymphocyte-adhesion moieties; and (iv)lymphocyte-activating moieties, and optionally an immune stimulant.

BACKGROUND OF THE DISCLOSURE

Some cancers, such as advanced pancreatic cancers, are un-resectable atthe time of their discovery. Additionally, cancer relapse followingsurgery remains a major clinical problem and is frequently the ultimatecause of death. Relapse often occurs because tumors cannot be completelyresected, as they invade vital organs and/or lack distinct borders. Toeradicate residual tumor, transfusions of tumor-reactive lymphocytes,referred to as adoptive cell therapy (ACT), are currently being testedin cancer patients as one of the most promising treatment options.However, two major hurdles remain that seriously limit the use of ACT toprevent tumor relapse: infused lymphocytes inefficiently traffic totumor, and even if a limited number of administered lymphocytesinfiltrate tumor tissue, they poorly persist. Accordingly, although somepatients benefit enormously from ACT, in most cases the tumor willultimately grow back, with lethal consequences.

SUMMARY OF THE DISCLOSURE

The present disclosure provides compositions comprising (i) a structure;(ii) lymphocytes, (iii) lymphocyte-adhesion moieties; and (iv)lymphocyte-activating moieties, and optionally an immune stimulant. Thecompositions can be surgically implanted at a site of an un-resectabletumor or at a tumor resection site following the resection. Suchcompositions can act as active depots, releasing lymphocytes into thetumor area or resection bed to purge residual tumor cells. In someembodiments, at the same time, dispersed immune stimulants can activatethe subject's immune system to destroy distant deposits of tumor cells.Supporting components of the compositions, includinglymphocyte-activating moieties assist with lymphocyte multiplication andactivation while lymphocyte-adhesion moieties assist with lymphocytemovement out of the composition into the tumor cell area. Thus, thecompositions can provide a clinical device to provide surgeons with amore effective treatment option for tumors that currently cannot beresected or can only be managed by palliative surgery.

Treatment with the compositions and methods disclosed herein can savepatients from complicated second or third surgeries, costly extendedhospital stays, rounds of radiation or chemotherapy, and expensivepalliative care.

Thus, disclosed herein is a composition comprising (i) a structurecomprising an injectable polymer or scaffold comprising pores; (ii)lymphocytes disposed within the structure, (iii) at least onelymphocyte-adhesion moiety associated with the structure; and (iv) atleast one lymphocyte-activating moiety associated with the structure.

In another embodiment, the lymphocytes are T-cells and/or natural killercells. In another embodiment, the lymphocytes are CD8+ T-cells. In yetanother embodiment, the composition comprises at least 7×10⁶lymphocytes.

In another embodiment, the lymphocyte-adhesion moiety comprises acollagen-mimetic peptide, a peptide that binds α₁β₁ integrin, α₂β₁integrin, α₄β₁ integrin, α₅β₁ integrin, or lymphocyte functionassociated antigen (LFA-1), a GFOGER (SEQ ID NO:1)_peptide, an ICAM-1peptide, or a FNIII₇₋₁₀ peptide. In yet another embodiment, thelymphocyte-adhesion moiety comprises a peptide of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, or SEQ ID NO:4.

In another embodiment, the lymphocyte-activating moieties are bound toor incorporated in one or more particles, wherein the particles aremicroparticles or nanoparticles. In another embodiment, the particlesare microparticles with a diameter of 10-20 μm and the ratio ofmicroparticles to lymphocytes within the composition is 0.5:1; 1:1; 5:1or 10:1. In yet another embodiment, the particles are nanoparticles witha diameter of 100-150 nm and the ratio of nanoparticles to lymphocyteswithin the composition is 500:1; 1000:1 or 5000:1. In anotherembodiment, the composition comprises 7×10⁶ to 1×10¹⁰ particles,

In another embodiment, lymphocyte-activating moiety comprises antibodiesspecific for CD3, CD28, and/or CD137.

In another embodiment, the composition further comprises an immunestimulant. In yet another embodiment, the particles further comprise animmune stimulant. In another embodiment, the immune stimulant is acytokine, an antibody, a small molecule, an siRNA, a plasmid DNA, and/ora vaccine adjuvant. In another embodiment, the cytokine is IL-2, IL-4,IL-10, IL-11, IL-12, IL-15, IL-18, TNFα, IFN-α, IFN-8, IFN-γ, or GM-CSF.In another embodiment, the immune stimulant is the interleukin-15superagonist RLI. In yet another embodiment, the immune stimulant is avaccine adjuvant such as CpG oligodeoxynucleotide or Poly(I:C).

In another embodiment, the structure is injectable.

In yet another embodiment, the lymphocyte-adhesion moieties and/orlymphocyte-activating moieties are associated with the structure in abioactive coating on the scaffold. In another embodiment, thelymphocyte-activating moieties are associated with particles embedded inthe pores of the scaffold. In yet another embodiment, thelymphocyte-activating moieties are associated with particles attached tothe surface of the scaffold or are embedded in the scaffold. In anotherembodiment, the scaffold is an alginate scaffold. In yet anotherembodiment, the scaffold is a polymeric calcium cross-linked alginatescaffold.

In yet another embodiment of the composition, the lymphocytes,lymphocyte-adhesion moieties, and lymphocyte-activating moieties arewithin the structure of the composition.

Also disclosed herein is a method of treating a tumor in a subjectcomprising implanting a composition of any one of Exemplary Embodiments1-33 into a subject within a proximity to a tumor cell sufficient tolead to the destruction of the tumor cell in the subject, therebytreating the tumor.

Further disclosed herein is a method of reducing surgical treatmentfailure caused by metastatic relapse after resection of a primary tumor,comprising administering a composition of any one of ExemplaryEmbodiments 1-33 to a tumor resection bed of a subject thereby reducingsurgical treatment failure caused by metastatic relapse after primarytumor resection.

In another embodiment, the implanting is within a tumor resection bed.In another embodiment, the implanting leads to the destruction of atumor cell of an incompletely resected tumor or a tumor cell of ametastasized tumor.

In another embodiment, the destroyed tumor cell is a cell of anincompletely resected tumor. In yet another embodiment, the destroyedtumor cell is a cell of a metastasized tumor.

In another embodiment, the tumor cell is a seminoma cell, a melanomacell, a teratoma cell, a neuroblastoma cell, a glioma cell, a rectalcancer cell, an endometrial cancer cell, a kidney cancer cell, anadrenal cancer cell, a thyroid cancer cell, a skin cancer cell, a braincancer cell, a cervical cancer cell, an intestinal cancer cell, a livercancer cell, a colon cancer cell, a stomach cancer cell, a head and neckcancer cell, a gastrointestinal cancer cell, a lymph node cancer cell,an esophageal cancer cell, a colorectal cancer cell, a pancreatic cancercell, an ear, nose and throat (ENT) cancer cell, a breast cancer cell, aprostate cancer cell, a uterine cancer cell, an ovarian cancer cell, ora lung cancer cell. In yet another embodiment, the tumor cell is aglioblastoma cell, a pancreatic adenocarcinoma cell or an ovarian cancercell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Delivery of tumor-reactive lymphocytes.

FIG. 2. Lymphocyte delivery approach using a composition disclosedherein. (1) biopolymer scaffold, (2) lymphocyte seeding, (3) mousemammary tumor, (4) surgical resection, (5) resection cavity withresidual tumor, (6, 7) implantation of implant harboring tumor-reactivelymphocytes, (8) wound closure, (9) controlled release of tumor-fightinglymphocytes into the resection cavity and tumor-draining lymph nodes.

FIGS. 3A-3C. Rapid tumor cell clearance and systemic anti-tumor activitywith a disclosed composition (FIG. 3A) Biopolymer scaffold loaded withtumor-reactive T-cells immediately after implantation onto tumorresection bed. (FIG. 3B) Stimulatory microparticles trigger theexpansion of loaded T-cells within the interior pore spaces of thescaffold. Tumor-reactive T-cells are released into the surroundingtissue to destroy residual tumor. Free tumor antigen is taken up byantigen presenting cells (APCs). (FIG. 3C) Immune stimulants releasedfrom the scaffold activate and mature APCs to prime and expand tumorantigen-specific lymphocytes in the host to destroy distant metastasesthroughout the body.

FIGS. 4A-4D. Fully functional tumor-reactive CD8+ T-cells can beisolated and expanded from vaccine-immunized mice. (FIG. 4A) Schematicof the 4T1 tumor vaccine (4T1-STIM). The surface expression of thecostimulatory ligands B7.1 and 4-1BBL was confirmed by flow cytometry in(FIG. 4B). (FIG. 4C) Flow cytometric measurement of IFN-γ in lymphocytesharvested from immunized mice before (left panel) and after a seven dayin vitro expansion (right panel) on irradiated 4T1-STIM monolayer. (FIG.4D) ⁵¹Cr release assay of expanded CD8-purified lymphocytes targeting4T1 tumor or B16F10 control tumor.

FIGS. 5A, 5B. Intravenous or intracavitary bolus injections of tumorreactive T-cells fail to clear incompletely resected tumor. (FIG. 5A)Longitudinal bioluminescence imaging of Gau-luc-expressing 4T1 tumors.Bioluminescent tumor signal quantified per animal every two days over aperiod of 42 days. Representative images for day 12 (before and aftertumor resection) and day 30 (tumor relapse) are shown in the upperpanel. (FIG. 5B) Sequential bioluminescence imaging of adoptivelytransferred 4T1 tumor-reactive T-cells retrovirally transduced withCBR-luc. Representative images from a total of 5 mice/group imaged everytwo days are shown. T-cells were injected as bolus intravenously or intothe resection bed.

FIGS. 6A-6E. Migration and sustained viability of T-cells insidealginate scaffolds coated with a collagen-mimetic peptide. (FIG. 6A)Reaction scheme of GFOGER (SEQ ID NO:1) peptide (in the FIGs. andexperimental examples, SEQ ID NO: 2) coupling to alginate. (FIG. 6B)Fluorescence quantification of the DYLIGHT® 650-labelled GFOGER (SEQ IDNO:1) peptide in alginate scaffolds. Representative images of uncoatedversus peptide-coupled alginate discs are shown on the right. (FIG. 6C)Photomicrograph of a T-cell loaded alginate scaffold. Time-lapse imagesof T-cells migrating through unmodified or GFOGER (SEQ ID NO:1) peptidefunctionalized alginate scaffolds. A 10-fold magnified image is shown inthe inset to illustrate avid pore-to-pore migration of T-cells.Trajectories of individual T-cells tracked for two hours are shown inthe lower panels. Time averaged velocities with and without GFOGER (SEQID NO: 1) peptide are graphed in (FIG. 6D). (FIG. 6E) Percentages ofviable (Annexin-V/PI double-negative) T-cells following alginase enzymedigestion of scaffolds with and without GFOGER (SEQ ID NO: 1) peptide torecover T-cells.

FIGS. 7A-7E. GFOGER (SEQ ID NO:1) peptide-coated alginate scaffoldsdisperse functional T-cells into tissue. (FIG. 7A) Schematic diagram ofthe in vitro assay to quantify the migration of tumor-reactive T-cellsfrom an alginate scaffold into a tissue mimetic (3D fibrillar collagengel). (FIG. 7B) Light microscope images of 4T1 tumor-reactive CD8+T-cells that have migrated from GFOGER (SEQ ID NO:1) peptide-coatedscaffolds into 3D collagen gels. Scale bar: 100 μm. (FIG. 7C)Quantification of T-cells in the alginate scaffold and in the collagenmatrix. At indicated time points, T-cells were recovered from scaffoldsand collagen gel by alginase or collagenase enzyme digestion,respectively. The number of viable T-cells was determined by Trypan Blueexclusion and graphed. (FIG. 7D) ⁵¹Cr release assay of T-cells recoveredfrom collagen gel after 48 hours targeting 4T1 tumor or B16F10 controltumor. (FIG. 7E) ELISA analysis of IL-2 (at 24 hours), IFN-γ, and TNF-α(at 48 hours) secreted by recovered T-cells seeded on an irradiated 4T1STIM tumor cell monolayer.

FIGS. 8A-8C. Stimulatory microparticles or nanoparticles integrated intothe 3D pore structure of GFOGER (SEQ ID NO:1) peptide-coated alginatescaffolds. (FIG. 8A) Photomicrograph of lyophilized scaffold. (FIG. 8B)Light microscopy image of alginate scaffold with incorporatedstimulatory (anti-CD3/CD28/CD137 antibody-coated)poly(lactic-co-glycolic acid) (PLGA) microparticles (black dots). Ahigher magnification confocal image of a single microparticle is shownin the right panel. (FIG. 8C) Confocal micrograph of alginate scaffoldwith integrated stimulatory lipid-enveloped nanoparticles. Acryo-transmission electron microscopy (TEM) image of lipid-coated PLGAnanoparticles is shown in the right panel.

FIGS. 9A-9C. Tumor-reactive T-cells exit scaffolds over time andinfiltrate surrounding tissue and tumor-draining lymph nodes. (FIG. 9A)Photomicrograph of GFOGER (SEQ ID NO:1) peptide-coated alginate scaffoldseeded with 4T1 tumor-reactive T-cells. The scaffold is being placedwhere a primary 4T1 tumor was just incompletely excised. FIG. 9BSequential bioluminescence imaging of implanted T-cells. Cells wereretrovirally transduced with clickbeetle red luciferase. Representativeacquisitions from a total of four mice imaged every two days are shown.(FIG. 9C) Confocal image of tumor-reactive T-cells (labeled withCellTracker™ Green, Life Technologies) as they exit the alginatescaffold (Alexa-647-labelled) to populate the tumor resection bed fourdays after implantation.

FIGS. 10A-10D. Functional recombinant IL-15-superagonist RLI can beproduced in 293-F cells and efficiently encapsulated into PLGAparticles. (FIG. 10A) Three-dimensional model of the RLI fusion protein(adjusted from Mortier, J Biol Chem, 281, 1612-1619 (2006)). (FIG. 10B)SDS polyacrylamide gel stained with Coomassie blue showing purified RLIprotein (molecular weight: 34 kDa). (FIG. 10C) Carboxyfluoresceinsuccinimidyl ester (CFSE) dilutions of 4T1 tumor-specific CD8+ T-cellsafter a 6-day coculture on irradiated 4T1 tumor monolayers with orwithout 10 ng/ml exogenous RLI. (FIG. 10D) In vitro release kinetics ofRLI from PLGA micro- or nanoparticles in RPMI medium containing 10% FCSat 37° C. determined by ELISA. The RLI encapsulation efficiencies formicro and nanoparticles were 41% (±6%) and 37% (±8%), respectively.

FIGS. 11A, 11B. Stimulatory signals trigger sustained T-cell expansion.(FIG. 11A) Image of prosurvival cytokine, stimulatory antibodies,mesoporous silica microparticle, and lipid envelope. (FIG. 11B) Graphsshowing T-cell proliferation and T-cell migration into surroundingtissue for both plain scaffold (upper panels) and scaffold withmicroparticles (lower panels).

FIGS. 12A-12F. Longitudinal bioluminescence imaging ofluciferase-expressing 4T1 breast tumor tumors. (FIG. 12A) Bioluminescenttumor signal quantified per animal at 0, 6, 14, and 18 days.Luciferase-tagged tumor cells were transplanted into the mammary gland,and ten days later, tumors were resected in a way such that ˜1% residualdiseased tissue remained. Four different treatment groups were studied(10 mice/group); no T cells (control mice left untreated after surgery),intravenous, intracacity, or scaffold delivered 4T1 tumor-reactiveT-cells. (FIG. 12B) Bioluminescent tumor signal quantified per animalevery six days over a period of 30 days. (FIG. 12C) Survival of animalsfollowing T-cell therapy illustrated by Kaplan-Meier curves. (FIG. 12D)Sequential bioluminescence imaging of adoptively transferred 4T1tumor-reactive T-cell retrovirally transduced with luciferase. (FIG.12E) Bioluminescent T-cell signal quantified per animal every two daysover a period of 12 days. (FIG. 12F) Confocal image of tumor-reactiveT-cell (labeled with CellTracker™ Green (Thermo Fischer Scientific,Inc.)) as they exit the scaffold (Alexa-647-labeled) to populate thetumor resection bed four days after implantation.

FIG. 13. Sequential bioluminescence imaging of adoptively transferred4T1 tumor-reactive T-cells retrovirally transduced with CBR-luc at 0, 4,8 and 12 days after administration. Representative images from a totalof 3 mice/group imaged every four days are shown.

FIG. 14. Polypeptide sequence of the GFOGER (SEQ ID NO:1) peptideadhesion motif.

FIG. 15. Polypeptide sequence of the GFOGER (SEQ ID NO:1) peptide usedin the Examples (SEQ ID NO. 2).

FIG. 16. Polypeptide sequence of the ICAM-1 cell adhesion molecule (SEQID NO. 3).

FIG. 17. Polypeptide sequence of the FN-III₇₋₁₀ fragment (SEQ ID NO. 4).

FIGS. 18A-18G. (FIG. 18A) Photomicrograph of a T-cell loaded alginatescaffold. Time-lapse images of T-cells migrating through unmodified orCollagen mimetic peptide (in the FIGs. and experimental examples theGFOGER (SEQ ID NO:1)-peptide SEQ ID NO: 2 is used) coated alginatescaffolds. Shown are trajectories of individual T-cells tracked for 30minutes. (FIG. 18B) Graph showing mean displacements of T-cells duringthe 30 minute imaging interval. (FIG. 18C) Schematic diagram of the invitro assay to quantify the migration of tumor-reactive T-cells from analginate scaffold into a tissue mimetic (3D collagen gel). Lightmicroscope images of tumor-reactive T-cells that have migrated from thescaffold into the 3D collagen gel are shown in the lower panel. (FIG.18D) Quantification of T-cells in the alginate scaffold and in thecollagen matrix. At indicated time points, T-cells were recovered fromscaffolds and collagen gel by alginase or collagenase enzyme digestion,respectively. The number of viable T-cells was determined by Trypan Blueexclusion and graphed. (FIG. 18E) Light microscopy image of alginatescaffold with incorporated microspheres. Particles were created bycoating porous silica microparticles with lipid bilayers that mimic cellmembranes. The high pore volume and surface area of the silica coreallow high-capacity encapsulation and sustained release of solublebiomolecules. The T-cell stimulant interleukin 15 superagonist, which isan interleukin 15 (IL-15)/IL-15Rα fusion protein that exhibits 50-foldgreater potency than IL-15 alone was encapsulated. The lipid membraneused to envelop particles serves as a modular scaffold for theattachment of a variety of lymphocyte-stimulating ligands. Agonisticanti-CD3, anti-CD28, and anti-CD137 monoclonal antibodies werecovalently coupled to the surface of microspheres containingIL-15/IL-15Rα. These prepared particles were then added to a GFOGER (SEQID NO:1) peptide-modified alginate solution before molding 3D scaffolds.(FIG. 18F) Quantification of T-cell egress from plain scaffolds, versusscaffolds carrying stimulatory microparticles. Using the in vitro assayfrom FIGS. 18C and 18D, the number of viable T-cells in the scaffold andthe surrounding collagen gel at given time points was determined. (FIG.18G) CFSE dilutions of T-cells embedded in plain versusmicroparticle-functionalized scaffolds were analyzed by flow cytometry 7days after cell seeding.

DETAILED DESCRIPTION

A projected 1.5 million new patients will be diagnosed with solid tumorsin the United States in 2013. Most of them will undergo surgery, basedon the premise that a major resection leads to longer survival.Nonetheless, surgery is often considered a palliative venture with nohope of cure, as many tumors infiltrate vital organs or criticalstructures that cannot be resected. Adjuvant chemotherapy and radiationtreatments can increase the duration of survival, but in most cases arenot curative interventions. While monoclonal antibodies havesignificantly improved the outcome of patients undergoing surgery, mostpatients succumb to disease relapse. Hence, no adjuvant therapy iscurrently available that can reliably eradicate all microscopic residualdisease following solid-tumor surgery.

An ideal anti-tumor immunotherapy should not only eradicate residualtumor cells quickly to prevent tumor relapse, but should also activatethe patient's own immune system to induce systemic anti-tumor memory forcontrol of metastatic tumors and long-term tumor resistance. In theory,cancer vaccines are capable of eliciting such anti-tumor activity,however in reality, residual tumors progress very rapidly and literallyoutpace the immune system. Conversely, tumor-reactive T-cells infused inACT can lyse large tumor deposits immediately, yet the majority oftransferred cells never reach their intended target or are rendereddysfunctional by the tumor microenvironment. Another limitationcurrently restricting the widespread use of ACT is the need to growsufficient numbers of tumor-reactive cells in the laboratory (10¹⁰-10¹¹cells/patient). This is an extremely costly, laborious process performedin a few locations worldwide.

Disclosed herein are compositions and methods that can treatun-resectable tumors or non-resected tumor cells and therefore treatcancer, metastasis, and/or tumor relapse. The compositions include astructure, lymphocytes, lymphocyte-adhesion moieties andlymphocyte-activating moieties. Lymphocyte-adhesion moieties assist withlymphocyte exit from the structure following implantation at a treatmentsite. Lymphocyte-activating moieties support activation, multiplicationand/or maintenance of the lymphocytes within the structure.

The structure can be an injectable structure or a scaffold with pores.In embodiments including a scaffold, the pores can provide a structureto embed tumor-targeting lymphocytes. In some embodiments, the scaffoldis formed from a material having lymphocyte-adhesion moieties. In otherembodiments, lymphocyte-adhesion moieties can be provided as part of abioactive coating that fully or partially coats the scaffold.

When a scaffold is used, lymphocyte-activating moieties can be a part ofthe scaffold itself, can be provided as part of a bioactive coatingand/or can be provided on particles. The described particles can bemicro- or nanoparticles and can be embedded in the pores of thescaffold, attached to the surface of the scaffold and/or be embeddedwithin the scaffold itself. The composition may further comprise one ormore immune stimulants.

The described compositions and methods provide surgeons with a powerfultool to effectively deliver and functionally support tumor-reactivelymphocytes to target un-resectable tumors and/or to purge healthytissue and lymph nodes of residual tumor cells following a resection.The compositions sustain the viability of, stimulate the proliferationof, and/or amplify the anti-tumor activity of embedded lymphocytes. Thecompositions and methods also allow the controlled release oflymphocytes directly into the tumor or tumor bed following resection totreat cancer and/or to protect against disease metastasis and/orrecurrence.

The described compositions obviate the need to grow billions of cells inthe laboratory as all essential stimulatory lymphocyte-activatingmoieties for activation of embedded lymphocytes are incorporated intothe structure of the composition itself. Thus, only a small number(10⁶-10⁷) of minimally cultured lymphocytes are needed to prepare thedescribed compositions. Embedded lymphocytes then rapidly expand in situwithin the structure and migrate out of the composition with theassistance of lymphocyte-adhesion moieties. Already at the treatmentsite, the lymphocytes immediately begin eliminating tumor cellsincluding residual tumor cells in a resection bed. As used herein, theterms “resection bed” and “tumor resection bed” refer to the areaimmediately surrounding the previously resected tumor. The smallernumber of required lymphocytes to effectuate the treatments allows manycancer centers, where even only rudimentary cell-processing facilitiesare available, to take advantage of the treatment benefits offered bythe currently disclosed compositions and methods.

In particular embodiments, the compositions of the current disclosurecan deliver tumor-reactive lymphocytes (FIG. 3A) along with immunestimulants at high local concentrations and over an extended period oftime. Lymphocytes seeded within the composition exit the compositionfollowing implantation and disperse at high densities throughout a tumorresection bed and into draining lymph nodes to destroy remainingresidual tumor cells following a resection (FIG. 3B). This step releaseslarge amounts of tumor antigens from dying tumor cells into the tissue,which are subsequently taken up by antigen presenting cells (APCs). Atthis point, the compositions can play a second key role. By releasing apotent immune stimulant, they can activate APCs and tumor-reactiveimmune cells to mount a robust host anti-tumor immune response. This“second wave” of anti-tumor immunity is broader and involves multiplecell types (FIG. 3C) acting in synergy to eliminate remaining tumorcells. This described approach provides immediate efficacy byimplantation of anti-tumor lymphocytes. At the same time, thecomposition is designed to turn the tumor site or tumor resection cavityinto a “self” vaccine site using dying tumor cells directly as thesource of antigen to launch an effective anti-tumor immune response inthe host.

The described compositions also provide in vivo screening tools to (1)test which immune cell types, cell phenotypes or combinations of immunecells are most potent at destroying tumors, and (2) identify agents thatboost their anti-tumor activity. The conventional approach currentlyused to answer these critical questions usually involves intravenousinjection of the immune cells to be tested into tumor-bearing animals.However once infused, cells poorly traffic to the tumor and quicklychange their phenotype, which prevents the determination of theirintrinsic anti-tumor activity. Likewise, small molecules, cytokines orstimulatory antibodies are often rapidly cleared from the circulationfollowing intravenous administration and poorly penetrate into solidtumors. This makes it extremely difficult to study their direct effectson immune cells in tumors. Within the disclosed compositions, any immunecell type can be embedded into the compositions along with a giventherapeutic compound and, upon implantation, is directly exposed totumor under clinically relevant in vivo conditions. This approachprovides previously inaccessible knowledge about how immune cellscollaborate and how drugs affect their function, which could ultimatelyspeed up the transition of cell-based immunotherapies to the clinic.

Various components of the compositions and methods are now described inmore detail.

Polymers

The structures of the compositions can be constructed from a variety ofmaterial including, without limitation, biocompatible polymers.Exemplary biocompatible polymers include, but not limited to, agar,agarose, alginate, alginate/calcium phosphate cement (CPC),beta-galactosidase (β-GAL), (1,2,3,4,6-pentaacetyl a-D-galactose),cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acidcollagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate)(PHBHHx), poly(lactide), poly(caprolactone) (PCL),poly(lactide-co-glycolide) (PLG), polyethylene oxide (PEO),poly(lactic-co-glycolic acid) (PLGA), polypropylene oxide (PPO),poly(vinyl alcohol) (PVA), silk, soy protein, and soy protein isolate,alone or in combination with any other polymer composition, in anyconcentration and in any ratio. Blending different polymer types indifferent ratios using various grades can result in characteristics thatborrow from each of the contributing polymers. Various terminal groupchemistries can also be adopted.

When injectable structures are used, the polymers can be responsive to achanged environmental condition following implantation. Polymers withthese characteristics are known to those of ordinary skill in the art.For example, in one embodiment, an injectable in situ gel-forming systemis used. In one embodiment, the polymer formulation can gel in vivo inresponse to temperature change (thermal gelation), in response to pHchange or in response to light. For example, polymers that gel inresponse to ultraviolet (UV) light can be used. In another embodiment,the polymer formulation can gel in vivo in response to ioniccross-linking. In another embodiment, the polymer formulation can gel invivo in response to solvent exchange. In one embodiment, the gel used isthermoreversible, pH reversible, or light reversible. In anotherembodiment, the gel used is high-viscosity and shear-thinning. Inadditional gelling embodiments, the gel can be a gel formed from,without limitation, any polymer described herein.

In particular embodiments, alginate is used as a structure material,either separately or in combination with one or more other materials.Alginate is easily processed, water soluble, and non-immunogenic.Alginate is a biodegradable anionic polysaccharide with free hydroxylgroups that offer easy gelling. In alternative embodiments, the polymermay be a polyelectrolyte complex mixture (PEC) formed from a 1:1solution of alginate and chitosan.

In one embodiment, a structure may formed from an alginate/calciumcarbonate/glucono-delta-lactone mixture, such as 0.5-5% alginate, 0.5-15g/L calcium carbonate, and 1-50 g/L gluconon-delta-lactone in a ratio of2:1:1 (alginate:CaCO₃:GDL). Polymer structures may also include varyingamounts of gelatin in combination with varying amounts of alginate.Depending on the materials and material ratios in mixture, thestructures may optionally be cross-linked.

In particular embodiments, polymer solutions having varying amounts ofpolymer dissolved in an acidic solution can be used to form thestructures disclosed herein. The concentration of the acid can beadjusted depending on the amount of polymer dissolved. In one aspect,the acidic solution is 1% (v/v) acetic acid. In one embodiment, theamount of polymer in solution is between about 0.5-5% (w/v) and anywhole or partial increments therebetween. For example, the amount ofpolymer in solution (w/v) can be 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%,4.5% or 5%. In one embodiment, the amount of polymer in solution is 2.4%(w/v). In other various embodiments, the polymer is dissolved in atleast one of water, acid, acetic acid, camphene, orcamphene-naphthalene.

When gelatin is incorporated, the concentration of the acid can beadjusted depending on the amount of gelatin in combination with polymer(in one embodiment, alginate) that is dissolved. In one aspect, theacidic solution is 1% (v/v) acetic acid. In one embodiment, the amountof gelatin in solution is between about 1-10% (w/v) and any whole orpartial increments therebetween. For example, the amount of alginate insolution (w/v) can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In oneembodiment, the amount of alginate in solution is 5.5% (w/v). In anotherembodiment, the polymer solution includes a combination of 2.4% (w/v)alginate solution and a 5.5% (w/v) gelatin solution. In other variousembodiments, the gelatin in combination with varying amounts of alginateis dissolved in at least one of water, acid, acetic acid, camphene, orcam phene-naphthalene.

In another embodiment, alginate-based scaffolds can be formed asfollows: a weight by volume (w/v) alginate solution in deionized (DI)water can be prepared and filtered with a 0.45 micrometer bottle filterto remove any particles and then frozen to −80° C. The frozen sample canbe lyophilized in a 4.5 liter benchtop freeze dry system (Labconco,Kansas City, Mo.). The filtered lyophilized alginate can bereconstituted into solutions of various concentrations (0.1%-5%) withwater or buffer.

Crosslinking can be performed with, without limitation, calcium chlorideand/or calcium carbonate. Calcium carbonate is a slow crosslinker, withsamples taking up to several hours to fully crosslink. To increase thespeed of the reaction gluconodeltalactone (GDL) can be added. Calciumchloride is a fast crosslinker and the samples will fully gel in a fewminutes. In one method, the addition of CaCl₂ to the alginate solutioncan occur prior to freezing. Other methods include use of a 5.5% (w/v)solution of calcium carbonate+GDL added to the alginate solution priorto initial freezing.

In particular embodiments, alginate solutions can be degassed in a speedmixer and poured slowly into casts to prevent bubbles from forming. Whenpipetting the polymer solutions into small molds, air bubble formationcan be avoided by placing a micropipette on the open end of mold groovesand repeatedly flushing the entire canal system until the residual airis flushed out.

Freeze casting can be used to form the scaffolds disclosed herein.Various polymer solutions can be freeze cast into various sized casts aswould be understood by those skilled in the art. The rate of coolingshould be controlled as it affects the size and alignment of pores, aswell as the formation of ridges. In one embodiment, the cooling rate canrange between 0.1-100° C. per minute (m) and any whole or partialincrements therebetween. In a preferred embodiment, the cooling rate canrange between 1−10° C./m, and any whole or partial incrementstherebetween. For example, the cooling rate (° C./m) can be 0.1, 0.5, 1,2, 3, 4, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

Lymphocyte-Adhesion Moieties

The compositions disclosed herein include lymphocyte-adhesion moietiesto promote lymphocyte mobility out of the implanted structures.Lymphocyte-adhesion moieties include, without limitation, cell-adhesionmoieties such as cell-adhesion polypeptides that mimic the extracellularmatrix (such as collagen). As used herein, “cell adhesion polypeptides”refer to compounds having at least two amino acids per molecule whichare capable of binding via cell surface molecules, such as integrin. Thecell adhesion polypeptides may be any of the proteins of theextracellular matrix which are known to play a role in cell adhesion,including fibronectin, vitronectin, laminin, elastin, fibrinogen,collagen types I, II, and V, as described in Boateng et al., Am. J.Physiol.—Cell Physio. 288:30-38 (2005), which is incorporated byreference herein for their teachings regarding the same. Additionally,the cell adhesion polypeptides may be any peptide derived from any ofthese proteins, including fragments or sequences containing the bindingdomains. Cell adhesion polypeptides include those havingintegrin-binding motifs, such as the ICAM-1 motif, and related peptidesthat are functional equivalents. Cell adhesion polypeptides may also beany of the peptides described in U.S. Patent Publication No. 20060067909which is incorporated by reference herein for its teachings regardingthe same.

In particular embodiments, the structures include compounds havinglymphocyte-adhesion moieties, such as a ligand for α₁β₁ integrin, aligand for α₂β₁ integrin, a ligand for α4β₁ integrin, a ligand for α₅β₁integrin, a ligand for lymphocyte function-associated antigen (LFA-1),or combinations thereof. In certain embodiments the ligand interactsspecifically with one integrin. In still other embodiments, the ligandis not a complete fibronectin molecule or is not a complete collagenmolecule.

The lymphocyte-adhesion moiety can be a peptide, antibody, or a smallorganic molecule. A small organic molecule refers to a carbon-basedmolecule having a molecular weight of 500 daltons or less. The antibodyor an integrin binding fragment thereof can be single chained,humanized, or chimeric. In certain embodiments, the lymphocyte-adhesionmoiety can be a collagen-mimetic peptide, for example a stabletriple-helical, collagen-mimetic peptide that contains the GFOGER (SEQID NO:1) adhesion motif from type I collagen that is recognized by theα₂β₁ integrin. This peptide adopts a stable triple-helical conformationsimilar to the native structure of type I collagen. An exemplarycollagen-mimetic peptide has the following amino acid sequenceGGYGGGPC(GPP)₅GFP*GER(GPP)₅GPC (SEQ ID NO: 2). In one embodiment, theGFOGER (SEQ ID NO:1) peptide comprises, consists, or consistsessentially of SEQ ID NO:2.

Another embodiment provides ICAM-1 as a lymphocyte-adhesion moiety.ICAM-1 is an Ig-like cell adhesion molecule that binds integrinspromoting cell-cell adhesion and is a ligand for lymphocytefunction-associated (LFA) antigens. ICAM-1 is found primarily onmonocytes and endothelial cells, and is widely inducible, orupregulated, on many cells including T-cells, B-cells, thymocytes,dendritic cells, endothelial cells, fibroblasts, keratinocytes,chondrocytes, and epithelial cells. This protein has a co-stimulatoryeffect upon cytotoxic T-cell interaction, and is utilized in a number ofintercellular binding interactions. In one embodiment, ICAM-1 comprises,consists, or consists essentially of SEQ ID NO:3.

Another embodiment provides FNIII₇₋₁₀ as a lymphocyte-adhesion moiety.FNIII₇₋₁₀ is a fibronectin fragment spanning the 7-10th type III repeatsof fibronectin. The sequence of fibronectin is known in the art. In oneembodiment, FNIII₇₋₁₀ comprises, consists or consists essentially of SEQID NO:4.

Effective variants of the sequences disclosed herein can also be used.Variants include peptides having one or more conservative amino acidsubstitutions. As used herein, a “conservative substitution” involves asubstitution of one amino acid for another found in one of the followingconservative substitutions groups: Group 1: Alanine (Ala), Glycine(Gly), Serine (Ser), Threonine (Thr); Group 2: Aspartic acid (Asp),Glutamic acid (Glu); Group 3: Asparagine (Asn), Glutamine (Gin); Group4: Arginine (Arg), Lysine (Lys), Histidine (His); Group 5: Isoleucine(Ile), Leucine (Leu), Methionine (Met), Valine (Val); and Group 6:Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp).

Additionally, amino acids can be grouped into conservative substitutiongroups by similar function or chemical structure or composition (e.g.,acidic, basic, aliphatic, aromatic, sulfur-containing). For example, analiphatic grouping may include, for purposes of substitution, Gly, Ala,Val, Leu, and Ile. Other groups containing amino acids that areconsidered conservative substitutions for one another include:sulfur-containing: Met and Cysteine (Cys); acidic: Asp, Glu, Asn, andGln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser,Thr, Pro, and Gly; polar, negatively charged residues and their amides:Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg,and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, andCys; and large aromatic residues: Phe, Tyr, and Trp. Additionalinformation is found in Creighton (1984) Proteins, W.H. Freeman andCompany which is incorporated by reference for its teachings regardingthe same.

Variants also include sequences with at least 70% sequence identity, 80%sequence identity, 85% sequence, 90% sequence identity, 95% sequenceidentity, 96% sequence identity, 97% sequence identity, 98% sequenceidentity, or 99% sequence identity to SEQ ID NO:1; SEQ ID NO:2; SEQ IDNO:3; and SEQ ID NO:4.

“% identity” refers to a relationship between two or more proteinsequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenproteins as determined by the match between strings of such sequences.“Identity” (often referred to as “similarity”) can be readily calculatedby known methods, including (but not limited to) those described in:Computational Molecular Biology (Lesk, A. M., ed.) Oxford UniversityPress, N Y (1988); Biocomputing: Informatics and Genome Projects (Smith,D. W., ed.) Academic Press, N Y (1994); Computer Analysis of SequenceData, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.)Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. andDevereux, J., eds.) Oxford University Press, NY (1992), eachincorporated by reference herein for its teachings regarding the same.Preferred methods to determine identity are designed to give the bestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the Lasergene bioinformatics computingsuite (DNASTAR®, Inc., Madison, Wis.). Multiple alignment of thesequences can also be performed using the Clustal method of alignment(Higgins and Sharp CABIOS, 5, 151-153 (1989), incorporated by referenceherein for its teaching regarding the same) with default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include theGCG suite of programs (Wisconsin Package Version 9.0, Genetics ComputerGroup (GCG), Madison, Wis.); BLASTP, BLASTN, BLASTX (Altschul, et al.,J. Mol. Biol. 215:403-410 (1990), incorporated by reference herein forits teaching regarding the same); DNASTAR®; and the FASTA programincorporating the Smith-Waterman algorithm (Pearson, Comput. MethodsGenome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20.Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y. incorporatedby reference herein for its teaching regarding the same). Within thecontext of this disclosure it will be understood that where sequenceanalysis software is used for analysis, the results of the analysis arebased on the “default values” of the program referenced. As used herein“default values” will mean any set of values or parameters whichoriginally load with the software when first initialized.

Within certain embodiments, the lymphocyte-adhesion moieties may beincorporated into the backbone of a polymer chain. For example, apolymer can be created containing YIGSR in the backbone of a polymer asdescribed in Jun et al., J. Biomaterials Sci., Polymer Ed. 15(1), 73-94(2004), which is incorporated by reference herein for its teachingsregarding the same. One of skill in the art could incorporate other celladhesion polypeptides into the backbone of alginate or other polymers.

In another embodiment, the lymphocyte-adhesion moieties may be graftedonto a polymer. In particular embodiments, the lymphocyte-adhesionmoieties are polypeptides that may be grafted onto polymers usingvarious methods known in the art. In one method, polymers having sidebranches containing reactive functional groups such as epoxide, halide,amine, alcohol, sulfonate, azido, anhydride, or carboxylic acid moietiescan be covalently linked to the amine terminus of the polypeptides viathe reactive side branches using conventional coupling techniques suchas carbodiimide reactions. For example, RGD (Arg-Gly-Asp)-containingpeptides have been grafted onto the backbone of polymers as described inLin, et al., J. Biomedical Materials Res, 28(3), 329-42 (1994) which isincorporated by reference herein for its teachings regarding the same.In another example, RGD-containing peptides have been grafted onto theside branches of polyethylene glycol based polymers, as described inHansson, et al., Biomaterials, 26, 861-872 (2005).

When scaffolds are used as a structure, the scaffold can comprise fibrinscaffolds, collagen scaffolds or fibrin and/or collagen scaffold blends(blended with, for example, alginate). Certain embodiments do notrequire bioactive coatings to support lymphocyte mobility. Similarly,injectable forms of the structures do not require bioactive coatings.

Scaffolds can also be coated with a bioactive coating comprising alymphocyte-activating moiety. In particular embodiments, the scaffold isat least partially coated with a bioactive coating comprising alymphocyte-adhesion moiety. The bioactive coating can be applied ontothe surface of the scaffold in various ways, including the use ofcoating methods that are known in the art. For example, the bioactivecoating may be sprayed onto the scaffold by a conventional electrostaticspraying process, resulting in charged droplets being deposited onto thesurface of the composition As the coating fluid dries, the bioactivecompound, for example, a polypeptide, remains adhered to the surface ofthe composition by inter-molecular bonding with the side-chain groups onthe polypeptides. The deposited polypeptide may form a monolayer on thesurface of the scaffolding.

In particular embodiments, the bioactive coating may be bonded to thesurface of a scaffold by any type of chemical or physical bonding means,including covalent, polar, ionic, coordinate, metallic, electrostatic,or intermolecular dipolar (including Van der Waals) bonds.

In one embodiment, the surface of the scaffold is coated with GFOGER(SEQ ID NO:1) peptide. As an example, the purified GFOGER (SEQ ID NO:1)peptide could be stored as a trifluoroacetic acid (TFA) salt andreconstituted to 10 mg/mL in 0.1% TFA and 0.01% sodium azide and storedat 4° C. prior to use. After the scaffolds are rinsed with ethanol toremove contaminants, cleaned in fresh ethanol, rinsed in ddH₂O, they canbe soaked in phosphate buffered saline (PBS). The GFOGER (SEQ ID NO:1)peptide can then be absorbed onto the scaffolds passively by incubatingthe scaffolds in a solution of GFOGER (SEQ ID NO:1) peptide in PBS.Prior to implantation, scaffolds could be rinsed in PBS to removeunbound GFOGER (SEQ ID NO:1) peptides.

In preparation of an alginate composition, an alginate/calciumcarbonate/glucono-delta-lactone mixture can be prepared by stirring,with concentrations ranging from 0.5-5 wt % alginate, 0.5-15 g/L calciumcarbonate, and 1-50 g/L glucono-delta-lactone in a volume ratio of 2:1:1(alginate:CaCO₃:GDL) as a “pre-gelling” process. In particularembodiments, the resulting mixture can be freeze cast (directionallyfrozen) at a constant cooling rate (0.1°/min-10°/min) until solid andlyophilized until dry. The dried compositions can be crosslinked in0.1-2.5 wt. % calcium chloride for 5-30 minutes and washed in HEPESbuffered saline prior to any further use of the scaffold. For placementof a bioactive coating, surfaces of the compositions can be coated inpolylysine or polyornithine (0.1-1.0 mg/ml for 3-10 minutes) followed bycoating in a GFOGER (SEQ ID NO:1) peptide (10 μg/ml-250 μg/ml for 30minutes-24 hours).

In preparation of a alginate-chitosan composition, an alginate-chitosanpolyelectrolyte complex (PEC) mixture can be prepared by sonicating orhomogenizing on ice in a range of 1:1 to 1:9 solutions (both ways) ofalginate (prepared in water) and chitosan (prepared in 1% acetic acid)and total polymer content ranging from 0.5%-5%. The pH of the resultingmixture can be adjusted with NaOH up to 10.0. In particular embodiments,the alginate-chitosan PEC mixture can be freeze cast at a constantcooling rate (0.1°/min-10°/min) until solid and lyophilized until dry.Dried compositions can be crosslinked in 0.1-2.5% calcium chloride for5-30 minutes and washed in PBS prior to any further use of thecomposition. For bioactive coating, scaffolds can be coated inpolylysine or polyornithine (0.5 mg/ml for 6 minutes) followed bycoating in a GFOGER (SEQ ID NO:1) peptide (10 μg/ml-250 μg/ml for 30minutes-24 hours).

In particular embodiments, GFOGER (SEQ ID NO:1) peptides are immobilizedonto alginate using aqueous carbodiimide chemistry.

Bioactive coatings can additionally include other components to alterthe surface of the scaffold, for example polylysine, polyornitine, orother glycoproteins.

Lymphocyte-Activating Moieties

Lymphocyte-activating moieties include any compound that activates alymphocyte and can be incorporated in or attached to the structuresdisclosed herein. As used herein, activation of a lymphocyte refers tothe state of a lymphocyte that has been sufficiently stimulated toinduce detectable cellular proliferation, cytokine production, oreffector function such as tumor targeting and/or killing. If thelymphocyte is a T-cell, activation also results in expression of cellsurface markers particular to the T-cell type. Exemplarylymphoctye-activating moieties include CD3, CD27, CD28, CD80, CD86,4-1BB, CD137, OX40, CD30, CD40, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3,and CD83 ligands or antibodies, CD1d, recombinant CD1d moleculespreloaded with α-galactosyl ceramide and/or recombinant majorhistocompatibility complex (MHC) molecules loaded with defined tumorantigens or peptides to selectively expand particular lymphocyte typesembedded within a scaffold.

Lymphocyte-activating moieties can be found within injectable forms ofthe structures or embedded within the pores of the scaffolds, attachedto the surface of the scaffolds, and/or embedded within the scaffoldsthemselves. As discussed further below, lymphocyte-activating moietiescan also be associated with particles.

Immune Stimulants

In particular embodiments, immune stimulants can be included within thecompositions. In certain embodiments, the immune stimulant is acytokine, an antibody, a small molecule, an siRNA, a plasmid DNA, and/ora vaccine adjuvant.

Exemplary cytokines include, without limitation, IL-2, IL-7, IL-12,IL-15, IL-18, IL-21, TNFα, IFN-α, IFN-β, IFN-γ, or GM-CSF. In anotherembodiment the immune stimulant may be a cytokine composition comprisingcombinations of cytokines, such as IL-2, IL-12 or IL-15 in combinationwith IFN-α, IFN-β or IFN-γ, or GM-CSF, or any effective combinationthereof, or any other effective combination of cytokines. Theabove-identified cytokines stimulate T_(H)1 responses, but cytokinesthat stimulate T_(H)2 responses may also be used, such as IL-4, IL-10,IL-11, or any effective combination thereof. Also, combinations ofcytokines that stimulate T_(H)1 responses along with cytokines thatstimulate T_(H)2 responses may be used.

Exemplary antibodies include, without limitation, anti-PD1, anti-PDL1,anti-CTLA-4, anti-TIM3, agonistic anti-CD40, agonistic anti-4-1BB,and/or bispecific antibodies (e.g., BITE-antibodies: anti-CD3/anti-tumorantigen). Exemplary small molecule drugs include, without limitation,TGF-beta inhibitors, SHP-inhibitors, STAT-3 inhibitors, and/or STAT-5inhibitors. Any siRNA capable of down-regulating immune-suppressivesignals or oncogenic pathways (such as kras) can be used whereas anyplasmid DNA (such as minicircle DNA) encoding immune-stimulatoryproteins can be used. Exemplary vaccine adjuvants, include, withoutlimitation, any kind of Toll-like receptor ligand or combinationsthereof (e.g. CpG, Poly(I:C), α-galactoceramide, MPLA, cyclicdinucleotides, VTX-2337 (novel TLR8 agonist developed by VentiRx),and/or inhibitors of heat-shock protein 90 (Hsp90), such as 17-DMAG(17-dimethylaminoethylamino-17-demethoxygeldanamycin).

Immune stimulants derived from the molecules noted in the precedingparagraphs can also be used. For example, RLI is an IL-15-IL-15receptor-α fusion protein that exhibits 50-fold greater potency thanIL-15 alone. IL-15 impacts the anti-tumor immune response at multiplepoints. It can differentiate monocytes into stimulatory antigenpresenting cells; promote the effector functions and proliferation oftumor-reactive T-cells; and recruit and activate NK cells.

Immune stimulants can be found within injectable forms of the structuresor embedded within the pores of the scaffolds, attached to the surfaceof the scaffolds and/or embedded within the scaffolds themselves. Asdiscussed further below, immune stimulants can also be associated withparticles.

Release of the immune stimulants from particles can be modified byincorporation of surfactants, detergents, complexing agents, internalphase viscosity enhancers, surface active molecules, co-solvents,chelators, stabilizers, derivatives of cellulose, polysorbates, PVA orsucrose. Salts and buffers can also be used to alter releasecharacteristics.

Particles

In particular embodiments, particles can be included as means todeliver/present lymphocyte-activating moieties to lymphocytes becausethey can mimic physiological antigen presenting cells. Another advantageof particles is that they are highly modular and can be customizedwithout affecting the chemical properties of the structure itself.

In particular embodiments, lymphocyte-activating moieties and/or immunestimulants are provided in association with particles. Particles can beincluded within injectable structures and/or within scaffolds. Theparticles can be formed from any biocompatible polymer including,without limitation, agar, agarose, alginate, alginate/CPC, β-GAL,(1,2,3,4,6-pentaacetyl a-D-galactose), cellulose, chitin, chitosan,collagen, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite,PHBHHx, poly(lactide), PCL, PLG, PEO, PLGA, PPO, PVA, silk, soy protein,and soy protein isolate, alone or in combination with any other polymercomposition, in any concentration and in any ratio. Blending differentpolymer types in different ratios using various grades can result incharacteristics that borrow from each of the contributing polymers.Various terminal group chemistries can also be adopted.

In particular embodiments, the particles can be microparticles ornanoparticles. Microparticles can have a diameter of 10-20 μm whilenanoparticles have a diameter of 100-150 nm.

Particles may be formed according to any method known to those ofordinary skill in the art. Common methods include, without limitation,spray-drying or emulsion.

In one embodiment, an organic phase of PLGA polymer anddioleoylphosphocholine (DOPC), dioleoylphosphoglycerol (DOPG) andmalemimide-phycoerythin (PE) lipids are emulsified in water, leading toself-assembled lipid coatings surrounding each particle.

Particles may also be formed using a Buchi-190 Mini Spray wherein 1-2%polymer can be spray dried and collected in a dry container. Theparticles can be stirred in 0.4% sodium hydroxide in ethanol solutionfor 15 minutes to 1 hour before washing in PBS. The particles can becoated in 0.1-1% alginate solution for 5-20 minutes before washing inwater, freezing, and lyophilization until dry. For covalentcrosslinking, the particles can be stirred in a 0.001%-1% genipinsolution prepared in PBS, or 0.001-25% glutaraldehyde solution for 0-48hours. Crosslinking is stopped by stirring the particles in a 10%glycine (prepared in PBS) solution for 30 minutes. The particles canalso be coated in 0.1-1% alginate solution for 5-20 minutes, or alginatesolution followed by 0.1-1% polyethylene glycol solution for 5-20minutes before washing in water, freezing, and lyophilizing until dry.

In another embodiment, alginate particles can be cross-linked asfollows. Alginate particles (collected dry or collected in calciumchloride) can be suspended in ethanol. Epichlorohydrin (1-25% v/v) canbe added to the particle mixture in ethanol. The mixture can besonicated or homogenized on ice while adding 1M-6M sodium hydroxide. Themixture can then be stirred at room temperature for 6-24 hours and thereaction can be stopped by adjusting the pH to 7 with 1M hydrochloricacid. The crosslinked particle can be washed in ethanol in decreasingconcentrations (e.g., 75%, 50%, 25%), followed by washing in water threetimes. Alternate covalent crosslinking can be performed by suspendingthe particles in methanol containing 1-25% glutaraldehyde and 0.05-5%hydrochloric acid, stirring for 0-48 hours. Remaining calcium chloridecan be removed by stirring microcapsules in 55 mM sodium citrate for 10minutes, followed by washing in water. Particles can also be coated herein A) 0.1-1% chitosan solution (prepared in 1% acetic acid) for 5-20minutes, B) the solution of (A) followed by 0.1-1% alginate solution for5-20 minutes; and/or C) the solution of (A) followed by 0.1-1%polyethylene glycol solution for 5-20 minutes. The additionalcrosslinking/coating steps are followed by washing in water, freezing,and lyophilizing until dry.

Lymphocyte-activating moieties can be incorporated on the surface of theparticles by soaking (either from a dry state or pre-hydrated in PBS) inthe desired lymphocyte-activating moiety solution (with or withoutstabilizers such as trehalose). In particular embodiments,concentrations and timing can range from 1 μg/ml to 1 g/ml for 15minutes to 24 hours before rinsing with PBS.

In one embodiment, the lymphocyte-activating moieties are antibodiesthat are mildly reduced with dithiothreitol (DTT) and covalently coupledto maleimide on the surface of the particles.

In one embodiment, to incorporate the particles into compositions, thelymphocyte-activating moiety-surface coated particles can be stirredinto the polymer mixture prior to freeze casting. In one embodiment, theprepared particles are added dropwise to a 2% aqueous GFOGER (SEQ IDNO:1)-peptide modified alginate solution before cross-linking alginatewith calcium chloride and molding three-dimensional scaffolds by freezedrying. This process yields compositions with 7×10⁶ antibody-coated PLGAmicroparticles that are homogenously dispersed within the scaffold'spore network.

In an additional example of forming particles, lipid stock solutions canbe prepared in chloroform. DOPC, DSPE-PEG(2000) maleimide, cholesteroland 18:1 PEG(2000) PE can be combined in a scintillation vial to attaina DOPC:DSPE-PEG(2000) maleimide:cholesterol:PEG(2000) with a PE massratio of 55:5:30:10 and 2.5 mg total lipid. Chloroform can be evaporatedand residual solvent removed.

A suspension of spherical silica gel can be prepared in PBS. Thesuspension can be combined with an immune stimulant. The suspension canbe gently agitated and diluted. The entire suspension can be added to abatch of lipid film. The mixture can be vortexed. The particles can becentrifuged and the supernatant can be removed. The pellet can be washedand redispersed.

The hinge-region disulfide bonds of anti-CD3, CD28, and CD137 can beselectively reduced as described by Kwong et al, Biomaterials 32, 5134(2011) which is incorporated by reference herein for its teachingsregarding the same. After removal of the reducing agent, themildly-reduced antibodies can be added to the maleimide-functionalizedparticles. The mixture can be vortexed briefly and the resultingantibody-labeled particles can be centrifuged and supernatant removed.The pellet can then be washed.

Lymphocytes

The structures of the compositions disclosed herein include embeddedlymphocytes. Any type of lymphocyte capable of targeting and killingtumor cells, targeting tumor cells for killing by other cell types, orotherwise mediating tumor cell killing can be used. The lymphocytes areautologous to the individual for whom the composition is administered.

Lymphocytes include T-cells, B cells and natural killer (NK) cells. Thecurrent disclosure focuses on the use of embedded T-cells, but othertypes of lymphocytes may be used as well, alone or in combination.

Several different subsets of T-cells have been discovered, each with adistinct function. T-cells include helper cells (CD4+ T-cells) andcytotoxic T-cells (CTLs, CD8+ T-cells) which comprise cytolytic T-cells.

T helper cells assist other white blood cells in immunologic processes,including maturation of B cells into plasma cells and activation ofcytotoxic T-cells and macrophages, among other functions. These cellsare also known as CD4+ T-cells because they express the CD4 protein ontheir surface. Helper T-cells become activated when they are presentedwith peptide antigens by MHC class II molecules that are expressed onthe surface of antigen presenting cells (APCs). Once activated, theydivide rapidly and secrete small proteins called cytokines that regulateor assist in the active immune response.

Cytotoxic T-cells destroy virally infected cells and tumor cells, andare also implicated in transplant rejection. These cells are also knownas CD8+ T-cells because they express the CD8 glycoprotein at theirsurface. These cells recognize their targets by binding to antigenassociated with MHC class I, which is present on the surface of nearlyevery cell of the body.

A majority of T-cells have a T-cell receptor (TCR) existing as a complexof several proteins. The actual TCR is composed of two separate peptidechains, which are produced from the independent T-cell receptor alphaand beta (TCRα and TCRβ) genes and are called α- and β-TCR chains.Gamma-delta (γΔ) T-cells represent a small subset of T-cells thatpossess a distinct TCR on their surface. However, in γΔ T-cells, the TCRis made up of one γ-chain and one Δ-chain. This group of T-cells is muchless common (2% of total T-cells) than the αβ T-cells.

“Central memory” T-cells (or “T_(CM)”), as used herein, refers to anantigen experienced CTL that expresses CD62L or CCR-7 and CD45RO on thesurface thereof, and does not express or has decreased expression ofCD45RA as compared to naive cells. In embodiments, central memory cellsare positive for expression of CD62L, CCR7, CD2S, CD127, CD45RO, andCD95, and have decreased expression of CD54RA as compared to naivecells.

“Effector memory” T-cell (or “TEM”), as used herein, refers to anantigen experienced T-cell that does not express or has decreasedexpression of CD62L on the surface thereof as compared to central memorycells, and does not express or has decreased expression of CD45RA ascompared to a naive cell. In embodiments, effector memory cells arenegative for expression of CD62L and CCR7, compared to naive cells orcentral memory cells, and have variable expression of CD28 and CD45RA.

“Naive” T-cells, as used herein, refers to a non-antigen experienced Tlymphocyte that expresses CD62L and CD45RA, and does not express CD45ROas compared to central or effector memory cells. In some embodiments,naive CD8+ T lymphocytes are characterized by the expression ofphenotypic markers of naive T-cells including CD62L, CCR7, CD28, CD127,and CD45RA.

“Effector” or “TE” T-cells, as used herein, refers to a antigenexperienced cytotoxic T lymphocyte cells that do not express or havedecreased expression of CD62L, CCR7, CD28, and are positive for granzymeB and perforin as compared to central memory or naive T-cells.

Each of the lymphocytes types described herein can be embedded in thecompositions disclosed herein. In particular embodiments, the primarylymphocyte cell type will be CTL. CTLs can be included at 50% or more ofthe embedded lymphocyte population, 55% or more of the embeddedlymphocyte population, 60% or more of the embedded lymphocytepopulation, 65% or more of the embedded lymphocyte population, 70% ormore of the embedded lymphocyte population, 75% or more of the embeddedlymphocyte population, 80% or more of the embedded lymphocytepopulation, 85% or more of the embedded lymphocyte population, 90% ormore of the embedded lymphocyte population, 95% or more of the embeddedlymphocyte population, or 100% the embedded lymphocyte population.

Various combinations of lymphocytes can also be used in the compositionsdisclosed herein. In one embodiment, the composition includes a mixtureof CD8+ cells, NK cells, invariant NKT cells (iNKT cells), Th17 CD4+cells and/or B cells. In another embodiment, the compositions include amixture of CD8+ cells and NK cells. In another embodiment, the mixtureof CD8+ cells and NK cells is a 50:50 mix. In another embodiment, thecompositions include a mixture of CD8+ cells and iNKT cells. In anotherembodiment, the mixture of CD8+ cells and iNKT cells is a 50:50 mix. Allother possible combinations of the disclosed cell types can also be usedwithin the compositions disclosed herein.

In particular embodiments, the lymphocytes can be isolated and expandedfrom resected tumor. In another embodiment, subjects can be vaccinatedwith a tumor antigen (e.g., against Her2) and vaccine-induced T-cellpopulations can be expanded and embedded into the composition.

Lymphocytes within the compositions can be non-genetically modified orgenetically-modified or can be provided in a combination ofnon-genetically-modified and genetically-modified forms. Geneticmodifications can be made to enhance growth, survival, immune functionand/or tumor cell targeting. Examples of genetic modifications includethose allowing expression of a chimeric antigen receptor (CAR), a αβT-cell receptor (or modification thereof), and/or pro-inflammatorycytokines. CAR modification and/or αβ T-cell receptor modificationsallow the modified lymphocytes to specifically target cell types.

In one aspect, modified lymphocytes can have improved tumor recognition,trigger increased native T-cell proliferation and/or cytokineproduction. Different potential CAR nucleic acid constructs that encodedifferent ligand binding domains, different spacer region lengths,different intracellular binding domains and/or different transmembranedomains, can be tested in vivo (in an animal model) and/or in vitro toidentify CARs with improved function over non-genetically modifiedlymphocytes and/or other CARs and in particular embodiments, using thecompositions disclosed herein as an in vivo screening tool.

Exemplary CARs express ligand binding domains targeting, withoutlimitation, mesothelin, Her2, WT-1 and/or EGRF. An exemplary T-cellreceptor modification targets melanoma-associated antigen (MAGE) A3 TCR.

In some embodiments it may be desired to introduce functional genes intothe lymphocytes to allow for negative selection in vivo as described by,for example, Lupton et al., Mol. and Cell Biol., 11:6 (1991); andRiddell et al., Human Gene Therapy 3:319-338 (1992); see also thepublications PCT/US91/08442 and PCT/US94/05601 by Lupton et. al.describing the use of bifunctional selectable fusion genes derived fromfusing a dominant positive selectable marker with a negative selectablemarker and which are incorporated by reference herein for all theydisclose regarding selectable genes. This can be carried out inaccordance with known techniques (see, e.g., U.S. Pat. No. 6,040,177 atcolumns 14-17) or variations thereof that will be apparent to thoseskilled in the art based upon the present disclosure. For example, it iscontemplated that overexpression of a stimulatory factor (for example, alymphokine or a cytokine) may be toxic to the treated subject.Therefore, it is within the scope of the disclosure to include genesegments that cause the cells of the disclosure to be susceptible tonegative selection in vivo. By “negative selection” is meant that theinfused cell can be eliminated as a result of a change in the in vivocondition of the individual. The negative selectable phenotype mayresult from the insertion of a gene that confers sensitivity to anadministered agent, for example, a compound. Negative selectable genesare known in the art, and include, inter alia the following: the Herpessimplex virus type I thymidine kinase (HSV-I TK) gene, which confersganciclovir sensitivity; the cellular hypoxanthinephosphribosyltransferase (HPRT) gene, the cellular adeninephosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase.

Desired genes can be introduced into the lymphocytes prior to embeddingin a composition disclosed herein. Such introduction can be carried outby any method known in the art, including but not limited totransfection, electroporation, microinjection, lipofection, calciumphosphate mediated transfection, infection with a viral or bacteriophagevector containing the gene sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, sheroplast fusion, etc.Numerous techniques are known in the art for the introduction of foreigngenes into cells (see e.g., Loeffler and Behr, Meth. Enzymol, 217,599-618 (1993); Cohen et al., Meth. Enzymol, 217, 618-644 (1993); Cline,Pharmac. Ther, 29, 69-92 (1985)) and may be used in accordance with thepresent disclosure, provided that the necessary developmental andphysiological functions of the lymphocytes are not disrupted. In oneembodiment, the technique provides for the stable transfer of the geneto the cell, so that the gene is expressible by the cell and preferablyheritable and expressible by its cell progeny. In other embodiments, thetechnique provides for transient expression of the gene within a cell.

Methods commonly known in the art of recombinant DNA technology whichcan be used to genetically modify the lymphocytes are described inAusubel et al. (eds.), 1993, Current Protocols in Molecular Biology,John Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and Expression,A Laboratory Manual, Stockton Press, NY, both of which are incorporatedby reference herein for their relevant teachings.

In particular embodiments, lymphocytes will be embedded within thecompositions at or near the time of composition implantation in asubject, for example within 48 hours of implantation, within 36 hours ofimplantation, within 24 hours of implantation, within 12 hours ofimplantation, within 6 hours of implantation, within 3 hours ofimplantation, within 1 hour of implantation or within 30 minutes ofimplantation. Generally, lymphocyte loading into pre-molded scaffoldswill occur within 30 minutes of implantation whereas the loading willmore often occur closer (i.e., within 5 minutes; within 2 minutes,within 1 minute or within 30 seconds) to the actual implantation timewhen injectable forms of the compositions are used.

The lymphocytes can be fresh lymphocytes or can be previouslycryo-preserved lymphocytes. If previously-cryopreserved lymphocytes areused, they should be thawed quickly (e.g., in a water bath maintained at37°-41° C.) and chilled immediately upon thawing. It may be desirable tofurther treat the lymphocytes in order to prevent cellular clumping uponthawing. To prevent clumping, various procedures can be used, includingbut not limited to, the addition before and/or after freezing of DNase,low molecular weight dextran and citrate, hydroxyethyl starch, etc.Where necessary due to potential cytotoxicities, cryoprotective agentsshould be removed. After removal of cryoprotective agents, whennecessary, cell count and/or viability testing can be performed.

A variety of methods to embed the lymphocytes into structures disclosedherein can be used (“embedding” is also referred to as “seeding”). Forexample, passive (static) seeding can be used. In one embodiment,lymphocytes are resuspended in cell culture medium (e.g., RPMI). Thiscell suspension is then added dropwise on top of a lyophilized scaffold.In another embodiment, where static seeding is used, a lymphocytesuspension is seeded onto a structure and afterwards incubated for acertain time in the absence of agitation before being exposed to dynamicculture conditions, for example into a spinner flask that is slowlyagitated. In another embodiment, dynamic seeding can be used. Fordynamic seeding the structure and the lymphocyte suspension can beplaced together in, e.g., a container and the container is thenincubated with gentle agitation for a certain time allowing thelymphocytes to embed themselves within the structure. In additionalembodiments, rotational systems (including centrifuges) and/or vacuumsystems can be used. In additional embodiments, sheet-based lymphocyteseeding, electrostatic lymphocyte seeding, magnetic lymphocyte seeding,filtration lymphocyte seeding, and/or oscillating perfusion lymphocyteseeding can be used. Various combinations, such as, and withoutlimitation, rotational vacuum seeding can also be used. The use ofvarious biological hydrogels is also appropriate. For discussions of thevarious seeding options, see Li et al., Biotechnol. Prog, 17, 935-944(2001); Wendt et al., Biotechnology and Bioengineering, 84, 205-214(2003); Yang, et al., J. Biomed. Mater. Res, 55, 379-386 (2001); andSittinger et al., Int. J. Artif. Organs, 20, 57 (1997) each of which isincorporated by reference herein for its relevant teachings regardingthe same.

Methods of Use

The compositions described herein can be placed in the vicinity ofun-resecatable tumor and/or non-resected tumor cells to have ananti-tumor effect in a subject. As used herein, the terms “subject” or“individual” typically refer to a mammal, such as a human, but can alsobe another mammal such as, but not limited to, dogs, cats, rabbits,cows, horses, etc. A “tumor” is a swelling or lesion formed by anabnormal growth of cells (called neoplastic cells or tumor cells). A“tumor cell” is an abnormal cell that divides by a rapid, uncontrolledcellular proliferation and continues to divide after the stimuli thatinitiated the new division cease. Tumors show partial or complete lackof structural organization and functional coordination with the normaltissue, and usually form a distinct mass of tissue, which may be eitherbenign, pre-malignant or malignant.

As used herein, an anti-tumor effect refers to a biological effect,which can be manifested by a decrease in tumor volume, a decrease in thenumber of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or a decrease of various physiologicalsymptoms associated with the cancerous condition. An anti-tumor effectcan also be manifested by a decrease in recurrence or an increase in thetime before recurrence. Accordingly, the compositions disclosed hereincan be used to treat a variety of cancers, can prevent or significantlydelay metastasis, and/or can prevent or significantly delay relapse.

Cancer (medical term: malignant neoplasm) refers to a class of diseasesin which a group of cells display uncontrolled growth (division beyondthe normal limits), invasion (intrusion on and destruction of adjacenttissues), and sometimes metastasis. “Metastasis” refers to the spread ofcancer cells from their original site of proliferation to another partof the body. The formation of metastasis is a very complex process anddepends on detachment of malignant cells from the primary tumor,invasion of the extracellular matrix, penetration of the endothelialbasement membranes to enter the body cavity and vessels, and then, afterbeing transported by the blood, infiltration of target organs. Finally,the growth of a new tumor, i.e. a secondary tumor or metastatic tumor,at the target site depends on angiogenesis. Tumor metastasis oftenoccurs even after the removal of the primary tumor because tumor cellsor components may remain and develop metastatic potential.

Cancers that can be treated with the anti-tumor effects of thecompositions and methods disclosed herein include, without limitation,seminomas, melanomas, teratomas, neuroblastomas, gliomas, rectal cancer,endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, skincancer, cancer of the brain, cervical cancer, intestinal cancer, livercancer, colon cancer, stomach cancer, head and neck cancer,gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectalcancer, pancreas cancer, ear, nose and throat (ENT) cancer, breastcancer, prostate cancer, cancer of the uterus, ovarian cancer, lungcancer, and metastases thereof.

Without limiting the scope of the compositions and methods disclosedherein, the following cancer types are noted:

Brain tumor (Glioblastoma): An estimated 10,000 new cases/year in theU.S. are seen. Currently no curative therapy is available. Gliobastomashows very infiltrative growth and cannot be resected completely. 90% oftumors relapse within a 2 cm margin from the originally resected tumor.Biomaterial wafers loaded with chemotherapy are United States Food andDrug Administration (FDA)-approved (GLIADEL®, MGI Pharma.) forglioblastoma. However, due to insufficient tissue penetration,biomaterial implant delivered chemotherapy is mostly ineffective. Incontrast, tumor-reactive lymphocytes deployed from the compositionsdisclosed herein can actively migrate to affected tissue, seeking outand destroying residual tumor cells.

Pancreatic adenocarcinoma: An estimated 43,920 new cases of pancreaticcancer were expected to occur in the U.S. in 2012. Only 20% will haveresectable disease at the time of diagnosis (80% of patients do notundergo surgery as their tumor is too advanced at the time ofdiagnosis). Even surgery is considered a palliative venture with a5-year survival rate of only 20%. Local recurrence is usually attributedto the difficulty of achieving microscopically negative surgicalmargins. Beyond the current composition's primary application toeradicate residual disease following surgical tumor resection, thecompositions could also provide pancreatic tumor patients withinoperable disease (˜80% of patients) with a highly effective treatmentoption. In this embodiment, compositions are implanted directly ontoun-resectable established pancreatic adenocarcinomas.

Ovarian cancer: An estimated 22,000 new cases in 2012 in the U.S. wereseen. Despite multimodality therapy with surgery and chemotherapy, mostovarian cancer patients have a poor prognosis (15,500 estimateddeaths/year in U.S.). Ovarian cancer primarily disseminates within theperitoneal cavity. Adoptive T-cell therapy in ovarian cancer patients iscurrently being investigated at several centers. However, to dateclinical results have been disappointing due to a poor survival ofinfused T-cells and a failure to combat immunosuppressive factorsreleased by tumor cells to render T-cells dysfunctional. Multiplecompositions embedded with tumor-reactive lymphocytes could be implantedlaparoscopically into the peritoneal cavity of ovarian cancer patients,where they release tumor-reactive lymphocytes and immune stimulants overan extended time period.

As will be understood by one of ordinary skill in the art, thecompositions are implanted in close proximity to un-resectable tumorcells and/or in tumor resection beds following resection. Thecompositions can be available in a number of different sizes and shapesand can be shape-conformable to fit the particular needs of individualsubjects. In particular embodiments, the compositions are injected usingultrasound guidance in close proximity to (or in physical contact with)un-resected or non-resected tumor cells. Depending on the stage, size orseverity of a tumor, compositions may be provided with differenttherapeutic strengths. Therapeutic strength can be manipulated byaltering the size of the composition, volume of the composition, thenumber of lymphocytes embedded within a composition, the number oflymphocyte-activating moieties within a composition, the presence oramount of immune stimulants within the composition, etc. Each of theseparameters can be assessed and determined by a treating physician.

For the purposes of the present disclosure, the term “proximity” refersto a distance within 10 cm, within 9 cm, within 8 cm, within 7 cm,within 6 cm, within 5 cm, within 4 cm, within 3 cm, within 2 cm, within1 cm, within 0.9 cm, within 0.8 cm, within 0.7 cm, within 0.6 cm, within0.5 cm, within 0.4 cm, within 0.3 cm, within 0.2 cm, or within 0.1 cm ofan un-resectable tumor, un-resectable tumor cells, and/or a tumorresection bed.

It is also understood by one of ordinary skill in the art that thecompositions can be implanted only once, at the time of resection or ata first treatment time in an individual with an un-resectable tumor.Additionally, the compositions can be implanted a plurality of times toprovide ongoing therapy over months or years. Such treatment regimenscan be determined by a treating physician.

As used herein, the term “surgical treatment failure” refers to relapseof cancer in a subject who had previously undergone tumor resection.Surgical treatment failure may include metastatic relapse.

EXAMPLES

The Examples below are included to demonstrate particular embodiments ofthe disclosure. Those of ordinary skill in the art should recognize inlight of the present disclosure that many changes can be made to thespecific embodiments disclosed herein and still obtain a like or similarresult without departing from the spirit and scope of the disclosure.

Example 1. Generation and Ex Vivo Expansion of 4T1 Breast Tumor-ReactiveMouse T-Cells

To model clinical ACT, in which tumor-reactive T-cells are isolated frompatients and expanded in the laboratory, an established protocol toobtain breast tumor-specific T-cells from BALB/c mice (Restifo, Nat RevImmunol 12, 269-281 (2012) which is incorporated by reference herein forits teachings regarding the same) was optimized. First, a 4T1 mammarycarcinoma cell line that expresses the costimulatory ligands B7.1 and4-1 BBL was generated using retroviral vectors (FIGS. 4A and 4B). Thisgenetic modification helps the immune system recognize 4T1 tumorantigens as foreign. Irradiated 4T1 B7.1/4-1 BBL (hereafter 4T1-STIM)cells act as a whole cell cancer vaccine and prime tumor-specificT-cells in BALB/c mice following tail-base injection (4×10⁶ tumorcells). To enhance further vaccine-driven immune responses, theadjuvants CpG oligodeoxynucleotide and poly(I:C) were mixed with4T1-STIM cells before injection. Following a prime-boost immunization,3×10⁵ 4 T1 tumor-reactive T-cells from inguinal and axillary lymph nodesof a single mouse can routinely be isolated. This is equivalent to 2% ofall cells in the lymph node, as determined by flow cytometricmeasurement of IFN-γ after a 12-hour restimulation on 4T1-STIM cells(FIG. 4C, left panel). Subsequently, 4T1-specific CD8+ T-cells wererapidly expanded ˜40-fold by in vitro coculture on 4T1-STIM monolayersin the presence of IL-2 and IL-15. The generated T-cells are functional(FIG. 4C, right panel) and selectively lyse 4T1 tumor cells (FIG. 4D).

Example 2. Tumor-Reactive T-Cells Injected Intravenously or Locally intothe Tumor Resection Bed Fail to Prevent Tumor Relapse Due to InefficientTumor Homing and/or Poor Persistence

Whether standard intravenous injections of 4T1 tumor-reactive T-cellscould reduce cancer relapse emanating from incompletely excised 4T1tumor was assessed. The chosen 4T1 breast tumor model very closelymimics the tumor growth and metastatic spread of human breast cancer tolymph nodes, liver, lung, and bone. Tumor cells, retrovirally taggedwith Gaussia luciferase for bioluminescence imaging, are easilytransplanted into the right mammary gland of BALB/c mice and developtumors that are ˜10 mm in size after two weeks. At that time point, micewere preconditioned for the adoptive transfer of T-cells by removinghomeostatic cytokine sinks by lymphodepletion (250 mg/kgcyclophosphamide, injected intraperitoneally). The following day, tumorswere resected, leaving behind 0.1-1% residual disease as quantified bybioluminescent imaging (FIG. 5A, “Residual tumor”). Mice were eitherinfused with T-cells the same day or received no treatment.Unexpectedly, all of the 10 animals treated intravenously with 7×10⁶tumor-specific T-cells relapsed with tumor (FIG. 5A, middle panel “Tumorrelapse”) and succumbed to disease at about the same time as untreatedcontrol animals (median survival: 33 versus 30 days, respectively;P=0.14). In an attempt to better protect mice from tumor recurrence,tumor-reactive T-cells were injected directly into the tumor resectioncavity during surgery. Such intracavitary T-cell administrations yieldeda statistically significant survival benefit compared with intravenouscell infusions (median survival: 37 versus 33 days, respectively;P=0.026). Nonetheless, residual tumor was not cleared in any of thetreated mice and quickly relapsed from the primary resection site andfrom tumor-draining lymph nodes (FIG. 5A, right panel).

Why T-cell treatments failed to control disease recurrence wasinvestigated. To track the in vivo migration and accumulation oftransferred T-cells in relation to residual 4T1 tumor, T-cells wereretrovirally-transduced with clickbeetle red luciferase (CBR-luc).Intravenously infused T-cells accumulated at high levels in the spleenand the liver, but poorly trafficked to relapsing tumor (FIG. 5B, upperpanel). T-cells injected directly into the tumor bed cavity were readilydetectable using bioluminescence on day 0. However, serial imagingshowed a gradual CBR-luc signal decline following T-cell injection,consistent with poor T-cell expansion and persistence (FIG. 5B, lowerpanel).

Taken together, these results suggest that, despite the high tumor celllysis observed in cytotoxicity assays in vitro (FIG. 4D), bolusinjections of tumor-reactive T-cells given by the intravenous orintracavitary routes fail to control tumor relapse. This failure is dueto inefficient accumulation of injected T-cells at the tumor site and/ora poor T-cell persistence and proliferation in the tumor resection bed.

Example 3. Porous Polysaccharide Scaffolds Coated with Collagen-MimeticPeptide Support Rapid “Lymph Nodelike” Motility and Sustain theViability of Embedded T-Cells

To address the issues noted above, implantable compositions were createdto produce a new microenvironment at the tumor resection site conduciveto the sustained proliferation of transferred lymphocytes. Thecompositions can be used to deliver tumor-reactive lymphocytes toresidual tumor following resection while sustaining their effectorfunction and survival. To function as a lymphocyte delivery and releaseplatform, a composition needs to provide sufficient mechanical supportfor embedded cells, a cell-adhesive coating to enable loaded cells tomigrate through the material and exit into tissue, and appropriatestimulatory signals to trigger cell proliferation.

As an example, under physiological conditions, T-cells migrate inperipheral tissue along collagen fibers. Whether anchoring thecollagen-mimetic GFOGER (SEQ ID NO:1) peptide to the inner walls ofporous alginate scaffolds could support intra-scaffold migration ofloaded T-cells was assessed. GFOGER (SEQ ID NO:1) peptide utilized inthe examples is a synthetic triple helical peptide (purchased from theMIT Biopolymers facility (SEQ ID NO:2)) that binds to the collagenreceptor α₂β₁ on T-cells. GFOGER (SEQ ID NO:1) peptide was immobilizedonto alginate using aqueous carbodiimide chemistry (FIG. 6A). Peptideswere fluorescently labeled with DYLIGHT® 650 to quantify couplingefficiencies using fluorescence imaging (FIG. 6B). Three-dimensionalscaffolds from calcium crosslinked alginate solutions by a freeze-drymethod were produced.

Peptide binding efficiencies ranged from 83% (0.005 mg peptide/mgalginate) for the lowest peptide concentration tested, to 53% (0.102 mgpeptide/mg alginate) for the highest peptide concentration tested (FIG.6B). The highest peptide concentration was selected for use insubsequent experiments.

To evaluate the effect of GFOGER (SEQ ID NO:1) peptide immobilization,T-cell migration inside scaffolds using time-lapse video microscopy wasrecorded (FIG. 6C). The GFOGER (SEQ ID NO:1) peptide coating supported arapid pore-to-pore T-cell migration quantitatively similar to the highmotility of these cells in native secondary lymphoid organs (7.9 μm/min,FIG. 6C). In contrast, T-cells poorly migrated through uncoatedscaffolds (3.6 μm/min), with most cells merely circling within theirinitial pore space (FIG. 6C, lower left panel). Notably, contact withGFOGER (SEQ ID NO:1) peptide sustained the viability of loaded T-cells(FIG. 6E), which is consistent with reports describing the activation ofpro-survival signaling pathways in T-cells binding collagen. The resultssuggest that GFOGER (SEQ ID NO:1) peptide-functionalized alginatescaffolds support rapid T-cell migration and sustain T-cell survival.

Example 4. Scaffolds Release Functional Tumor-Reactive T-Cells intoSurrounding Tissue

Whether T-cells can migrate outward from GFOGER (SEQ ID NO:1)peptide-coated scaffolds into surrounding tissue was examined. To mimiccollagen-rich and inflamed tissue near surgical resection margins,three-dimensional (3D) collagen gels containing the inflammatorycytokine IP-10 were prepared (FIG. 7A). Scaffolds loaded with 7×10⁶ 4 T1tumor-reactive T-cells were then embedded inside the collagen gel.T-cell egress from the composition was quantified every 12 hours forfour days by counting viable cells in the scaffold and the collagen gel.T-cells gradually populated the surrounding tissue mimetic at highnumbers, reaching a peak of 18.9×10⁶ T-cells after 72 hours (FIGS. 7B,7C). Due to the continuous proliferation of loaded effector T-cellswithin the pore space of scaffolds (FIG. 7B, lower panel), outward cellmigration only partially depleted the T-cell pool inside the scaffold(50% reduced T-cell number after four days, FIG. 7C).

To determine whether T-cells that migrated out of compositions werefunctional, their ability to lyse 4T1 tumor cells and to secretecytokines was measured. Composition-dispersed T-cells efficiently killed4T1 tumor targets and released high amounts of the effector cytokinesIL-2, IFN-γ, and TNF-α, following co-culture on 4T1-STIM cells (FIG.7E). In summary, GFOGER (SEQ ID NO:1)-peptide-coated scaffolds canefficiently disperse fully functional tumor-reactive T-cells intotissue.

Example 5. Incorporating Lymphocyte-Stimulating Ligands intoCompositions Will Help Delivered Lymphocytes Overcome Poor TumorImmunogenicity and an Adverse Tumor Microenvironment

Polymer microparticles coated with a combination of anti-CD3, CD28 andCD137 antibodies are incorporated into the compositions and supportproliferation of embedded lymphocytes and increase the number andfunctionality of the cells that migrate from the composition intosurrounding tissue.

Example 5a. Fabrication of Alginate Scaffolds Carrying StimulatoryMicro/Nanoparticles

To become activated, T-cells must not only recognize antigen but alsoreceive costimulatory signals from antigen presenting cells (APCs). Tomimic physiological T-cell stimulation inside the scaffold,poly(lactic-co-glycolic acid) (PLGA) microparticles similar in size toAPCs (10-20 μm in diameter, FIG. 8B, right panel) were synthesized.Particles were coated with avidin lipid so that biotinylated anti-CD3antibodies and co-stimulatory anti-CD28/CD137 antibodies could beanchored to their surface.

Stimulatory microparticle preparation (preparation of lipid film), Lipidstock solutions were prepared in chloroform. 140 μL DOPC (10 mg/mL), 30μL DSPE-PEG(2000) maleimide (5 mg/mL), 150 μL cholesterol (5 mg/mL), and50 μL 18:1 PEG(2000) PE (5 mg/mL) were combined in a scintillation vialto attain a DOPC:DSPE-PEG(2000) maleimide:cholesterol:PEG(2000) PE massratio of 55:5:30:10 and 2.5 mg total lipid. Chloroform was evaporatedunder a stream of nitrogen and residual solvent was removed under vacuumovernight.

Loading of Cytokine into Mesoporous Silica Microparticles.

A suspension of spherical silica gel (15 μm particle diameter, 100 Åpore diameter) was prepared in PBS (100 mg/mL). 120 μL of the suspensionwas combined in a 1.5-mL polypropylene tube with 400 μL of IL15 SA (22μg/m L). The suspension was gently agitated on a vortexer for 1 hour atroom temperature (RT) then diluted with 480 μL PBS.

Lipid Adsorption on Silica.

The entire SiO₂/IL15 suspension (1 mL) was added to a 2.5-mg batch oflipid film. The mixture was vortexed for 15 seconds at 10 minuteintervals for a total of 1 hour. The particles were centrifuged at3500×g for 2 minutes then the supernatant was removed. The pellet waswashed with PBS (3×1 mL) then redispersed in 500 μL PBS.

Antibody Conjugation to Silica-Supported Liposomes.

The hinge-region disulfide bonds of anti-CD3, CD28, and CD137 wereselectively reduced with dithiothreitol (DTT) as previously described[B. Kwong et al, Biomaterials (2011) 32:5134]. After removal of DTT witha desalting column, the mildly-reduced antibodies (anti-CD3: 200 μg;anti-CD28 and CD137: 400 μg) were added to the maleimide-functionalizedparticles. The mixture was vortexed briefly then rotated at RT for 2 h.The resulting antibody-labeled particles were centrifuged at 3500×g for2 min then the supernatant was removed. The pellet was washed with PBS(3×1 mL) then suspended in 125 μL PBS.

Prepared microparticles were then added dropwise to a 2% aqueous GFOGER(SEQ ID NO:1) peptide-modified alginate solution before cross-linkingalginate with calcium chloride and molding 3D scaffolds by freeze drying(FIG. 8A). 7×10⁶ antibody-coated PLGA microparticles were incorporatedinto a single alginate implant. Particles were homogeneously dispersedwithin the pore network of the scaffold (FIG. 8B, left panel).

To determine the impact of particle size on the capacity to stimulatelymphocytes inside compositions, in parallel experiments equal amountsof stimulatory antibodies from the surface of ˜100-fold smallernanoparticles (100-150 nm in diameter) are displayed. Lipid-envelopedPLGA nanoparticles are then fabricated. Steenblock, et al., J Biol Chem,286, 34883-34892 (2011) which is incorporated by reference herein forits teachings regarding the same. Briefly, an organic phase of PLGApolymer and DOPC, DOPG and maleimide-PE lipids are emulsified in water,leading to self-assembled lipid coatings surrounding each particle (FIG.8C, right panel). Anti-CD3/CD28/CD137 antibodies, mildly reduced withDTT, are then covalently coupled to maleimide on the surface ofnanoparticles as previously described. Bershteyn et al., Soft Matter, 4,1787-1791 (2008) which is incorporated by reference herein for itsteachings regarding the same. The amount of bioactive antibodies coupledto micro/nanoparticles is quantified by a functional ELISA assay. Thelipid envelopes of particles are first solubilized in 0.5% Tween 20surfactant.

Using recombinant mouse CD28/CD137/human Fc fusion proteins (R&DSystems) as capture agents, the amount of functional antibody instandard 96-well plates coated with anti-human IgG antibody usingHRP-conjugated detection antibodies is then measured. Particlesize/number is measured using the NanoSight LM20. Cryo-TEM images ofparticles (FIG. 8C, right) are generated by electron microscopy using aJEOL JEM 1400 Transmission Electron Microscope.

Example 5b. Comparison of Lymphocyte Expansion in Microparticle vs.Nanoparticle-Functionalized Compositions

The findings presented in FIGS. 8A-8C demonstrate that alginatescaffolds with incorporated stimulatory microparticles or nanoparticlescan be successfully fabricated. To identify the best candidatescaffold/particle composition, in vitro T-cell assays described indetail in FIGS. 7A-7E are conducted. 7 million 4T1 tumor-reactive CD8+T-cells are seeded either onto composite alginate/microparticlescaffolds (FIG. 8B), alginate/nanoparticle scaffolds (FIG. 8C) or plainscaffolds. For the initial studies, an equal number (7×10⁶) ofmicroparticles and T-cells are embedded in each scaffold. Depending onthe coupling efficiency of antibodies to the nanoparticles thiscorrelates to 1×10¹⁰ nanoparticles/scaffold. Both T-cell expansioninside the scaffolds and cell migration into surrounding tissue(collagen gel) over an 84-hour time period (FIG. 7C) are examined.Differences in the ability of scaffold-released T-cells to kill 4T1tumor (FIG. 7D) and to secrete effector cytokines (FIG. 7E) are alsoquantified.

Example 5c. Determination of Optimal Concentration of StimulatorySignals to Support Lymphocyte Expansion

The optimal concentration of stimulatory signals to support maximumlymphocyte expansion without compromising lymphocyte viability or keyeffector functions is determined. The main advantage of compositealginate/particle scaffolds is that the amount of stimulatory signalinside the scaffold can be finely tuned without further modifying thealginate backbone itself just by adding more or fewer stimulatoryparticles to the alginate solution before scaffold fabrication.Following identification of an optimized particle in Example 5b, theoptimal particle:lymphocyte ratio inside the composition is determined.Alginate/particle scaffolds with a final microparticle:T-cell ratio of0.5:1, 1:1, 5:1 and 10:1 (or 500:1, 1,000:1, 5,000:1 and 10,000:1, ifnanoparticles are chosen) are fabricated and compared to T-cellproliferation and functionality using the same assays described inExample 5b.

Before finalizing parameters regarding the optimal number of stimulatoryparticles to incorporate into compositions for all subsequent in vivostudies, the phenotype of composition-released lymphocytes ischaracterized. Phenotypic traits are predictive of the ability oflymphocytes to survive long-term, to serially kill tumor cells, tomigrate into tumor-draining lymph nodes and, in embodiments utilizingT-cells, to differentiate into memory T-cells. Therefore, in parallelstudies, how increasing stimulation inside the composition affects thephenotype of T-cells exiting the implant is investigated. T-cells thathave migrated out of alginate scaffolds (fabricated with various numbersof stimulatory particles as in previous experiments) into collagen gelafter 72 hours are recovered. T-cells are analyzed by flow cytometry forthe expression of the pro-survival factor Bcl-xL, the proliferationmarker Ki-67, the marker for terminal differentiation and replicativesenescence KLRG1, and the memory markers CD44, CD62L and CD122. Earlyapoptotic T-cells are identified by staining cells with Annexin-V andPI.

On the basis of pilot studies (FIGS. 7A-7E), comparative assays arecarried out using eight scaffolds/conditions. This sample size provides90% power to detect an effect size of 1 SD between groups, based on aone-way analysis of variance (ANOVA) with 2-sided significance level of0.05 (calculated with Prism 6.0 GraphPad software).

The major goal of providing lymphocytes with (co-)stimulatory signalsinside the composition is to compensate for the absence of these ligandson tumor cells—a mechanism used by tumors to render attackinglymphocytes dysfunctional. In particular, combined CD137 and CD28signaling can synergistically enhance the anti-tumor effector functionof T-cells while decreasing their susceptibility to apoptosis. Hence, itis expected that alginate scaffolds with incorporatedanti-CD3/CD28/CD137 antibodies will mount a robust proliferative T-cellresponse. However, functionally exhausting T-cells or causing activationinduced cell death by stimulating them excessively is avoided. If evenlow particle:T-cell ratios compromise T-cells functionality or survival,the strength of T-cell receptor activation is lowered. This is readilyachievable by reducing the number of anti-CD3 antibodies and increasingthe number of costimulatory anti-CD28/CD137 antibodies on the surface ofparticles.

Example 6. The Ability of Composition-Mediated Lymphocyte Delivery toPrevent Tumor Relapse More Effectively than Conventional LymphocyteInjections

The described studies in 4T1 breast tumor-bearing mice suggest thatneither systemic T-cell infusion nor local T-cell injection into thetumor resection cavity protects from disease recurrence (FIG. 5A). Thiswas in part due to a poor T-cell persistence and the inability of cellsto “find” residual tumor. To provide T-cells with niches that supporttheir function and stimulate their proliferation directly at theirprimary treatment site—the tumor resection bed—a porous materialcomposition delivery system was developed. Alginate matrices coated withthe collagen-mimetic GFOGER (SEQ ID NO:1) peptide sustain the viabilityof embedded T-cells (FIGS. 6A-6E) and disperse functional tumor-reactiveT-cells into surrounding tissue (FIGS. 7A-7E). Consistent with thisnotion, in mice T-cells exit implanted scaffolds at high densities overtime and infiltrate the tumor resection bed and tumor-draining lymphnodes (FIGS. 9A-9C). Based on this data, the objectives of Example 6 isto (1) compare the therapeutic effectiveness of composition-supportedlymphocyte delivery with conventional lymphocyte injections, and (2)elucidate underlying mechanism(s) by analyzing in vivo migration,expansion, persistence and phenotypic differences of transferredlymphocytes.

The studies will show that an appropriately designed composition canenhance the ability of lymphocytes to eradicate incompletely resectedtumor.

Example 6a. Comparison of the Therapeutic Effectiveness ofComposition-Supported Lymphocyte Delivery with Conventional LymphocyteInfections

Differences in the frequency of tumor relapse in BALB/c mice followingincomplete 4T1 tumor resection (as shown in FIGS. 2, 5A, 5B, 9A, 9B, and9C) are measured. Five different treatment groups are studied (18mice/group). In one group, 7×10⁶ 4 T1 tumor-reactive T-cells fromoptimized compositions are delivered directly into the tumor resectioncavity (as shown in FIG. 9A). Two groups of mice receive the same T-celldose, but cells are either injected intravenously or locally into theresection bed. To assess therapeutic effects of the biomaterial itself,“empty” scaffolds (no T-cells) are implanted into one additional groupof animals. All control mice are left untreated after surgery.

To quantitate differences in the tumor relapse rates between treatmentgroups, 4T1 tumors (retrovirally tagged with the Gaussia luciferasegene) are serially imaged every two days over a period of 42 days usingbioluminescence imaging, as described in relation to FIG. 5A) using astate-of-the-art IVIS Spectrum (Caliper/Xenogen) whole-mouse imagingsystem. On the basis of preliminary data (FIG. 5A), a whole animalbioluminescent signal of >40×10⁶ photons per second as the surrogateendpoint for death was defined to avoid unnecessary pain and distress intreated animals.

As described, alginate compositions are loaded with (co-)stimulatoryantibodies (FIGS. 8A, 8B), whereas T-cells injected as a cell suspensionare not supported by these ligands. Accordingly, composition-releasedT-cells would be expected to eradicate tumors more effectively merely asa result of receiving additional stimulation. Intravenously injectedT-cells therefore are activated locally with an equivalent amount ofstimulatory antibody incorporated into the compositions. To this end,T-cells are stimulated in 6-well plates with immobilizedanti-CD3/CD28/CD137 antibody 24 hours prior to injection.

Example 6b. Analysis of In Vivo Migration, Expansion, Persistence andPhenotypic Differences of Transferred Lymphocytes

Appropriate localization and migration of lymphocytes is a prerequisitefor anti-tumor responses. The described studies showed that dualbioluminescence imaging of Gaussia luciferase (Gau-luc) in 4T1 tumorcells and of clickbeetle red luciferase (CBR-luc) in tumor-reactiveT-cells allowed simultaneously monitoring of tumor regrowth and T-cellbiodistribution (FIGS. 5A, 5B). This assay is used to serially track thetissue distribution, expansion and persistence ofcomposition-administered lymphocytes. Animals are imaged every two daysfor a period of 42 days or until they need to be euthanized. Whetherthere is a relationship between lymphocyte localization and sites oftumor recurrence are examined. To this end, CBR-luc (T-cell) and Gau-luc(tumor cell) signal intensities over the areas of the tumor resectionsite and the axillary lymph nodes where distal metastases firstestablish (FIG. 5A) are quantified with data graphed as scatter plots toidentify correlations.

Example 6c. Effect of Delivery Mode on Phenotype or Functionality ofComposition-Administered Lymphocytes

Differences in lymphocyte phenotype and function between treatmentgroups are determined by flow cytometry. To distinguishcomposition-administered lymphocytes from host lymphocytes, 4T1tumor-specific T-cells are generated in BALB/c mice that are congenicfor the CD45.1 marker, as illustrated in FIG. 4A, and used to treatCD45.2 congenic recipient BALB/c mice. On day 4, 8 and 16 aftertransfer, three mice per treatment group are euthanized and single cellsuspensions prepared from the tumor-resection bed, tumor draining lymphnodes (inguinal, axillary), and the spleen. One fraction of cells isstained with antibodies against CD8, CD45.1 and CD107a (LAMP-1) todetect degranulation associated with cytotoxic target killing. Two othercell fractions re stained with the memory markers CD127, CD44 and CD62L.To compare functionality, an equal number of isolated lymphocytes arerestimulated for 12 hours on 4T1-STIM monolayers in the presence ofbrefeldin A, and intracellular IFN-γ or perforin are measured by flowcytometry, as shown in FIG. 4C. IFN-γ is a key effector cytokine, whileperforin is a key mediator of target cell killing.

The described experiments show that even “plain” GFOGER (SEQ ID NO:1)peptide-modified alginate scaffolds without stimulatory ligands sustainthe viability and the proliferation of transferred T-cells at a tumorresection site (FIG. 9B versus FIG. 5B). It is expected thatincorporating stimulatory cues into scaffolds will further enhance theexpansion and functionality of composition-delivered lymphocytes. Thistranslates into reduced tumor relapse rates in animals treated withcompositions disclosed herein versus T-cell injections.

Example 7. Co-Delivering Immune Stimulants from Implanted Compositionscan Enhance Act and Trigger Systemic Host Anti-Tumor Immunity

In Example 7, the ability of compositions, beyond their primary functionas lymphocyte delivery vehicles, to mount an effective host anti-tumorimmune response capable of eliminating untreated distant metastases aretested. RLI is an IL-15-IL-15 receptor a fusion protein (FIG. 10A) thatexhibits 50-fold greater potency than IL-15 alone. IL-15 impacts theanti-tumor immune response at multiple points. It can differentiatemonocytes into stimulatory antigen presenting cells; promote theeffector functions and proliferation of tumor-reactive T-cells; andrecruit and activate NK cells. However, like most potent immunestimulants, IL-15 or its superagonist RLI requires high and sustainedsystemic doses to achieve the desired effect, leading to dose-limitingtoxicities. Compositions disclosed herein focus drug action only onimmune cells for which they were intended, thereby avoiding systemicoverexposure to these agonists.

The experiments described in Example 7 investigate whether dispersingRLI from compositions into the tumor resection bed and the draininglymph nodes can (1) confer composition-delivered lymphocytes withmarkedly amplified anti-tumor effector functions and viability, and (2)orchestrate the destruction of untreated tumors throughout the body byactivating dendritic cells, T-cells and NK-cells in the host. Theresults will show that combining delivery of RLI and tumor-reactiveT-cells from compositions amplifies the expansion and persistence oftransferred T-cells, using low RLI doses that have no effect whenadministered by traditional systemic routes. The results will furthershow that composition-released RLI transforms the peritumoral tissue andthe tumor-draining lymph nodes from sites favoring immune suppressioninto “self” vaccine sites launching systemic anti-tumor immunity.

Example 7a

This example investigates whether dispersing RLI from implantedcompositions can amplify the effector function and persistence ofscaffold-delivered lymphocytes such as T-cells. 293-F cells aretransduced with a plasmid encoding His-tagged RLI protein and RLI ispurified from culture supernatant using a standard Cobalt agarose resin(FIG. 10B). Isolated RLI is fully functional, as demonstrated by itsability to enhance the proliferation of tumor-specific T-cellsco-cultured on tumor monolayers (FIG. 10C). Poly(lactic-co-glycolicacid) (PLGA) micro- or nanoparticles, fabricated as described in Example5a (including surface-anchored stimulatory antibodies), efficientlyencapsulate RLI and slowly release it over a one-week period (FIG. 10D).

Using the information from Example 5a as to optimized PLGA micro- ornanoparticle compositions (FIG. 8B), either RLI-loaded micro- ornanoparticles are loaded into alginate compositions. Biodistributions ofRLI following delivery via compositions disclosed herein relative tolocal or systemic bolus injections are measured. To this end, 10 μg RLIis administered either from compositions into 4T1 tumor resectioncavities, or it is injected in its soluble form intravenously or intoresection cavities. Blood serum and tissue samples of the tumorresection bed and tumor-draining (inguinal, axillary) lymph nodes recollected every two days for a period of 10 days. Tissue is homogenizedwith an ultrasonic dismembrator and centrifuged to collect supernatant.The amount of RLI per gram of tissue using a commercially available IL-15/IL-1 5Rα ELISA is measured.

Example 7b. Enhancement of Lymphocyte Function and Viability FollowingComposition-Released rli Versus Infusion-Administered RLI

One group of mice is treated with T-cell-loaded compositions thatcontain 10-50 μg RLI. A second group of mice is treated withT-cell-loaded compositions and 10-50 μg intravenous RLI. 4T1 tumorrelapse and T-cell expansion are quantitated in response to RLI givenvia different routes using bioluminescence imaging as in Example 2.

Example 7c. Composition-Released RLI Orchestration of Untreated and/orDistant Tumor

Tumors render antigen-presenting cells in draining lymph nodesdysfunctional to prevent tumor-specific T-cells in the host fromdifferentiating into cytolytic effectors. IL-15 has been reported torestore the antigen presenting capacity of dendritic cells (DCs) andreverse tolerance in tumor-specific T-cells. Thus, one predicted effectof local RLI delivery from compositions disclosed herein is activationof DCs in TDLNs coupled with the stimulation of anti-tumor T-cells inthe host.

The frequency and phenotype of DCs in the TDLNs (inguinal, axillary) inmice treated with RLI-loaded scaffold implants versus systemicallyinjected RLI (10 μg) or no exogenous RLI is analyzed. Lymph nodes andspleens are recovered from animals at day 2, 4 and 6 after treatment,digested with collagenase, and cells are stained with antibodies againstCD11c, CD11b, CD40, CD80, and MHC I and II to detect costimulatoryreceptors and MHC molecules by flow cytometry.

In situ cytokine induction in DCs is analyzed by intracellular IL-12p40staining. To measure the impact of RLI on the percentage oftumor-reactive T-cells in TDLNs, a fraction of cells on 4T1-STIMmonolayers is restimulated and analyzed for IFN-γ production, surfaceCD107a and perforin expression in CD8+ T-cells by flow cytometry.Another cell fraction is stained with antibodies against CD49b to assesswhether RLI increases the number of NK cells in the TDLNs or the spleen.

Example 7d. Ability of Compositions to Eliminate Distant TumorMetastases

The ultimate goal of local immunotherapy is the generation of a systemicimmune response capable of eliminating disseminated tumors and distantmetastases following treatment of an accessible tumor site. To testwhether local scaffold implantation into the tumor resection cavity candrive systemic/distal tumor inhibition, 4T1 tumors in the lungs of micethat just underwent incomplete 4T1 breast tumor resection areestablished. 4T1 tumor cells are known to form lung metastases wheninjected through the lateral tail vein. One million 4T1 tumor cellstagged with luciferase are infused to allow for bioluminescence imagingof 4T1 tumors in the lungs simultaneously with relapsing 4T1 tumors atthe primary tumor resection site. Five days after i.v. tumor injection,animals are treated with: (1) compositions loaded with 10-50 μg RLIonly, (2) compositions loaded with 7×10⁶ 4 T1 tumor-reactive CD8+T-cells only, (3) compositions loaded with both RLI plus T-cells, or (4)as control, “empty” (cell-free and RLI-free) compositions.

First differences in tumor growth between experimental groups at theresection site, the TDLNs and in the lungs using the bioluminescencetumor imaging assays described in Example 2 are quantitated. Toelucidate underlying mechanisms, tissue from the tumor resection cavity,the TDLNs, the lung, and the spleen are harvested at days 4, 8, and 12after treatment, and analyzed for frequencies and phenotypes oftumor-reactive T-cells, dendritic cells (DCs), and NK cells in the hostby flow cytometry as described above in relation to Examples 5c and 6b.To distinguish host cells from transferred cells, CD45.2⁺ recipient miceare treated with CD45.1⁺ T-cells.

For biodistribution studies and flow cytometry assays, 12 mice/conditionare studied (four mice/group, three experiments). For tumor imagingexperiments 18 mice/group are studied. Statistical analyses is performedas described in Example 5.

By exposing scaffold-deployed T-cells and host immune cells to highconcentrations of RLI over an extended period, synergistic anti-tumorresponses are elicited.

Example 8

Particles were created by coating porous silica microparticles withlipid bilayers that mimic cell membranes. Light microscopy image ofalginate scaffold with incorporated microspheres is shown in FIG. 18E.The high pore volume and surface area of the silica core allowhigh-capacity encapsulation and sustained release of solublebiomolecules. The T-cell stimulant interleukin 15 superagonist wasencapsulated. The lipid membrane used to envelop particles serves as amodular scaffold for the attachment of a variety oflymphocyte-stimulating ligands. Agonistic anti-CD3, anti-CD28 andanti-CD137 monoclonal antibodies were covalently coupled to the surfaceof microspheres containing IL-15/IL-15Rα. These prepared particles werethen added to a GFOGER (SEQ ID NO:1) peptide-modified alginate solutionbefore molding 3D scaffolds. An in vitro assay, a schematic of which isshown in FIG. 18C, is used to quantify the migration of tumor-reactiveT-cells from an alginate scaffold into a tissue mimetic (3D collagengel). Light microscope images of tumor-reactive T-cells that havemigrated from the scaffold into the 3D collagen gel are also shown inFIG. 18C.

A photomicrograph of a T-cell loaded alginate scaffold and time-lapseimages of T-cells migrating through unmodified or GFOGER (SEQ IDNO:1)-peptide-coated alginate scaffolds are shown in FIG. 18A. Thetrajectories of individual T-cells tracked for 30 minutes are shown.FIG. 18B shows a graph of mean displacements of T-cells during the30-minute imaging interval.

Quantification of T-cells in the alginate scaffold and in the collagenmatrix. At indicated time points, T-cells were recovered from scaffoldsand collagen gel by alginase or collagenase enzyme digestion,respectively. The number of viable T-cells was determined by Trypan Blueexclusion and graphed. (FIG. 18D).

Quantification of T-cell egress from plain scaffolds, versus scaffoldscarrying stimulatory microparticles. Using the in vitro assay from FIGS.18C,18D, the number of viable T-cells in the scaffold and thesurrounding collagen gel at given time points was determined. CFSEdilutions of T-cells embedded in plain versusmicroparticle-functionalized scaffolds were analyzed by flow cytometry 7days after cell seeding, the results of which are shown in FIG. 18G.

Example 9

Tumor cells were transplanted into the mammary gland, and ten dayslater, tumors were resected such that ˜1% residual diseased tissueremained. Four different treatment groups were compared. In one group,7×10⁶ 4 T1 breast tumor-specific T-cells contained in scaffold weredelivered directly into the tumor resection cavity. Two groups of micereceived lymphocytes injected intravenously or locally into theresection bed, and control mice were left untreated. Sequential in vivobioluminescence imaging of luciferase-expressing 4T1 breast tumors isshow in in FIG. 12A. Representative acquisitions from a total of 10mice/group imaged every two days are shown. Bioluminescent tumor signalquantified per animal every to days over a period of 30 days is shown inFIG. 12B. FIG. 19C shows survival of animals following T-cell therapyillustrated by Kaplan-Meier curves. Sequential bioluminescence imagingof adoptively transferred 4T1 tumor-reactive T-cell retrovirallytransduced with luciferase is shown in FIG. 12D. Bioluminescent T-cellsignal was quantified per animal every to days over a period of 12 days(FIG. 12E). FIG. 12F shows a confocal image of tumor-reactive T-cell(labeled with CellTracker Green) as they exit the scaffold(Alexa-647-labeled) to populate the tumor resection bed four days afterimplantation.

EXEMPLARY EMBODIMENTS

1. A composition comprising (i) a structure comprising an injectablepolymer or scaffold comprising pores; (ii) lymphocytes disposed withinthe structure, (iii) at least one lymphocyte-adhesion moiety associatedwith the structure; and (iv) at least one lymphocyte-activating moietyassociated with the structure.

2. A composition of embodiment 2, wherein the lymphocytes are T-cellsand/or natural killer cells.

3. A composition of embodiment 1 or 2, wherein the lymphocytes are CD8+T-cells. 4. A composition of any one of embodiments 1-3, comprising atleast 7×10⁶ lymphocytes.

5. A composition of any one of embodiments 1-4, wherein thelymphocyte-adhesion moiety comprises a collagen-mimetic peptide.

6. A composition of any one of embodiments 1-4, wherein thelymphocyte-adhesion moiety comprises a peptide that binds α₁β₁ integrin,α₂β₁ integrin, α₄β₁ integrin, α₅β₁ integrin, or lymphocyte functionassociated antigen (LFA-1).

7. A composition of any one of embodiments 1-4, wherein thelymphocyte-adhesion moiety comprises a GFOGER (SEQ ID NO:1) peptide.

8. A composition of embodiment 7, wherein the lymphocyte-adhesion moietycomprises a GFOGER (SEQ ID NO:1) peptide of SEQ ID NO:2.

9. A composition of any one of embodiments 1-4, wherein thelymphocyte-adhesion moiety comprises an ICAM-1 peptide.

10. A composition of embodiment 9, wherein the lymphocyte-adhesionmoiety comprises an ICAM-1 peptide of SEQ ID NO:3.

11. A composition of any one of embodiments 1-4, wherein thelymphocyte-adhesion moiety comprises a FNIII₇₋₁₀ peptide.

12. A composition of embodiment 11, wherein the lymphocyte-adhesionmoiety comprises a FNIII₇₋₁₀ peptide of SEQ ID NO. 4.

13. A composition of any one of embodiments 1-12, wherein thelymphocyte-activating moieties are bound to or incorporated in one ormore particles.

14. A composition of embodiment 13, wherein the particles aremicroparticles or nanoparticles. 15. A composition of embodiment 13 or14, wherein the particles are microparticles with a diameter of 10-20μm.

16. A composition of any one of embodiments 13-15,wherein the particlesare microparticles and the ratio of microparticles to lymphocytes withinthe composition is 0.5:1; 1:1; 5:1 or 10:1.

17. A composition of embodiment 13 or 14, wherein the particles arenanoparticles with a diameter of 100-150 nm.

18. A composition of any one of embodiments 13, 14 or 17, wherein theparticles are nanoparticles and the ratio of nanoparticles tolymphocytes within the composition is 500:1; 1000:1 or 5000:1.

19. A composition of any one of embodiments 1-18, wherein thelymphocyte-activating moiety comprises antibodies specific for CD3,CD28, and/or CD137.

20. A composition of any one of embodiments 13-19, wherein thecomposition comprises 7×10⁶ to 1×10¹⁰ particles.

21. A composition of any one of embodiments 1-21, further comprising animmune stimulant.

22. A composition of any one of embodiments 13-20, wherein the particlesfurther comprise an immune stimulant.

23. A composition of any one of embodiments 21 or 22, wherein the immunestimulant is a cytokine, an antibody, a small molecule, an siRNA, aplasmid DNA, and/or a vaccine adjuvant.

24. A composition of embodiment 23, wherein the cytokine is IL-2, IL-4,IL-10, IL-11, IL-12, IL-15, IL-18, TNFα, IFN-α, IFN-β, IFN-γ, or GM-CSF.

25. A composition of any one of embodiments 21-24, wherein the immunestimulant is the interleukin-15 superagonist RLI.

26. A composition of embodiment 23, wherein the vaccine adjuvant is CpGoligodeoxynucleotide or Poly(I:C).

27. A composition of any one of embodiments 1-26, wherein the structureis injectable.

28. A composition of embodiment 1, wherein the lymphocyte-adhesionmoieties and/or lymphocyte-activating moieties are associated with thestructure in a bioactive coating on the scaffold.

29. A composition of embodiment 1, wherein the lymphocyte-activatingmoieties are associated with particles embedded in the pores of thescaffold.

30. A composition of embodiments 28 or 29, wherein thelymphocyte-activating moieties are associated with particles attached tothe surface of the scaffold or are embedded in the scaffold.

31. A composition of embodiment 1, wherein the scaffold is an alginatescaffold.

32. A composition of embodiment 31, wherein the scaffold is a polymericcalcium cross-linked alginate scaffold.

33. A composition of any one of embodiments 1-32 wherein thelymphocytes, lymphocyte-adhesion moieties, and lymphocyte-activatingmoieties are within the structure of the composition.

34. A method of treating a tumor in a subject comprising implanting acomposition of any one of embodiments 1-33 into a subject within aproximity to a tumor cell sufficient to lead to the destruction of thetumor cell in the subject, thereby treating the tumor.

35. A method of embodiment 34, wherein the implanting is within a tumorresection bed.

36. A method of embodiment 34 or 35, wherein the destroyed tumor cell isa cell of an incompletely resected tumor.

37. A method of embodiment 34 or 35, wherein the destroyed tumor cell isa cell of a metastasized tumor.

38. A method of any one of embodiments 34-37, wherein the implantingleads to the destruction of a tumor cell of an incompletely resectedtumor or a tumor cell of a metastasized tumor.

39. A method of any one of embodiments 34-38 wherein the tumor cell is aseminoma cell, a melanoma cell, a teratoma cell, a neuroblastoma cell, aglioma cell, a rectal cancer cell, an endometrial cancer cell, a kidneycancer cell, an adrenal cancer cell, a thyroid cancer cell, a skincancer cell, a brain cancer cell, a cervical cancer cell, an intestinalcancer cell, a liver cancer cell, a colon cancer cell, a stomach cancercell, a head and neck cancer cell, a gastrointestinal cancer cell, alymph node cancer cell, an esophageal cancer cell, a colorectal cancercell, a pancreatic cancer cell, an ear, nose and throat (ENT) cancercell, a breast cancer cell, a prostate cancer cell, a uterine cancercell, an ovarian cancer cell, or a lung cancer cell.

40. A method of embodiment 39, wherein the tumor cell is a glioblastomacell, a pancreatic adenocarcinoma cell or an ovarian cancer cell.

41. A method of reducing surgical treatment failure caused by metastaticrelapse after resection of a primary tumor, comprising administering acomposition of any one of embodiments 1-33 to a tumor resection bed of asubject thereby reducing surgical treatment failure caused by metastaticrelapse after primary tumor resection.

42. A method of embodiment 41 wherein the primary tumor comprises aseminoma cell, a melanoma cell, a teratoma cell, a neuroblastoma cell, aglioma cell, a rectal cancer cell, an endometrial cancer cell, a kidneycancer cell, an adrenal cancer cell, a thyroid cancer cell, a skincancer cell, a brain cancer cell, a cervical cancer cell, an intestinalcancer cell, a liver cancer cell, a colon cancer cell, a stomach cancercell, a head and neck cancer cell, a gastrointestinal cancer cell, alymph node cancer cell, an esophageal cancer cell, a colorectal cancercell, a pancreatic cancer cell, an ear, nose and throat (ENT) cancercell, a breast cancer cell, a prostate cancer cell, a uterine cancercell, an ovarian cancer cell, or a lung cancer cell.

As will be understood by one of ordinary skill in the art, eachembodiment disclosed herein can comprise, consist essentially of orconsist of its particular stated element, step, ingredient or component.As used herein, the transition term “comprise” or “comprises” meansincludes, but is not limited to, and allows for the inclusion ofunspecified elements, steps, ingredients, or components, even in majoramounts. The transitional phrase “consisting of” excludes any element,step, ingredient or component not specified. The transition phrase“consisting essentially of” limits the scope of the embodiment to thespecified elements, steps, ingredients or components and to those thatdo not materially affect the embodiment. As used herein, a materialeffect would cause a statistically-significant reduction in theanti-tumor effects of a claimed composition or method in at least twomeasures of anti-tumor activity.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. When further clarity is required, the term “about” has themeaning reasonably ascribed to it by a person skilled in the art whenused in conjunction with a stated numerical value or range, i.e.denoting somewhat more or somewhat less than the stated value or range,to within a range of ±20% of the stated value; ±19% of the stated value;±18% of the stated value; ±17% of the stated value; ±16% of the statedvalue; ±15% of the stated value; ±14% of the stated value; ±13% of thestated value; ±12% of the stated value; ±11% of the stated value; ±10%of the stated value; ±9% of the stated value; ±8% of the stated value;±7% of the stated value; ±6% of the stated value; ±5% of the statedvalue; ±4% of the stated value; ±3% of the stated value; ±2% of thestated value; or ±1% of the stated value.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice.

Definitions and explanations used in the present disclosure are meantand intended to be controlling in any future construction unless clearlyand unambiguously modified in the following examples or when applicationof the meaning renders any construction meaningless or essentiallymeaningless. In cases where the construction of the term would render itmeaningless or essentially meaningless, the definition should be takenfrom Webster's Dictionary, 3^(rd) Edition or a dictionary known to thoseof ordinary skill in the art, such as the Oxford Dictionary ofBiochemistry and Molecular Biology (Ed. Anthony Smith, Oxford UniversityPress, Oxford, 2004).

What is claimed is:
 1. An alginate polymer scaffold coated with asynthetic collagen-mimetic peptide comprising SEQ ID NO: 2, wherein thescaffold houses: porous silica microparticles with a pore diameter ofabout 100 Å and wherein the microparticles are (a) coated with anti-CD3antibodies and anti-CD28 antibodies and (b) loaded with interleukin 15.2. A composition comprising (i) an injectable alginate polymer or animplantable alginate polymer scaffold comprising pores; and (ii) SEQ IDNO: 2, anti-CD3 antibodies, anti-CD28 antibodies, and interleukin
 15. 3.The composition of claim 2, further comprising lymphocytes.
 4. Thecomposition of claim 2, wherein the anti-CD3 antibodies and anti-CD28antibodies are bound to or incorporated in one or more particles.
 5. Thecomposition of claim 2, further comprising anti-CD137 antibodies.
 6. Thecomposition of claim 2, wherein the alginate polymer is injectable. 7.The composition of claim 2, wherein the alginate polymer is animplantable scaffold, and the SEQ ID NO: 2 is associated with thescaffold in a bioactive coating on the scaffold.
 8. The composition ofclaim 2, wherein the alginate polymer is an implantable scaffold, andthe anti-CD3 antibodies and anti-CD28 antibodies are associated withparticles embedded in the pores of the scaffold.
 9. The composition ofclaim 2, wherein the alginate polymer is an implantable scaffold and theanti-CD3 antibodies and anti-CD28 antibodies are associated withparticles attached to the surface of the scaffold or are embedded in thescaffold.